Novel cyanine compound for labeling biomolecule and preparation method thereof

ABSTRACT

Disclosed are a novel cyanine compound, represented by the following Formula 1, for labeling biomolecules, and a method for preparing the same. 
     
       
         
         
             
             
         
       
     
     wherein R 1 , R 2 , R 3 , R 4 , B, m and n are defined as above.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel cyanine compound forfluorescently labeling various biomolecules and a method for preparingthe same.

2. Description of the Related Art

Substitution groups in proteins that can be bound to reactive groups indyes can be inferred from the structure of amino acids (basic units ofproteins). For example, amino acid residues, more specifically, amino(—CH₂CH₂CH₂CH₂NH₂) for lysine, thiol (—CH₂SH) of cystein, imidazoleamine of histidine

secondary aliphatic hydroxyl group (—CH₂CH(OH)CH₃) of threonine, primaryaliphatic hydroxyl group (—CH₂OH) of serine and phenol hydroxyl group

of tyrosine and the like may be mentioned. Also, reactive groups in dyesmay be bound to n-terminal amino group (—COCHRNH₂) in amino acid. Inaddition, reactive groups in dyes may be bound to biomolecules such assugar, glycoprotein and antibodies.

Reactive groups used for dyes or molecules for labeling biomoleculesknown to date are classified depending on substitution groups bound tobiomolecules and are also called trivial names.

The most common reactive groups bound to amine of protein molecules areester and isothiocyanate, and the most common reactive group bound tothiol of protein molecules is maleimide and reactive groups bound tohydroxyl groups of protein molecules are as follows:

In addition to these reactive groups, numerous researchers andenterprises are designing reactive group intermediates exhibitingsuperior performance. Most intermediates exhibit short reaction timewith biomolecules and superior bonding performance, but are unstable inan aqueous solution state and are vulnerable to heat and produceby-products, while leaving groups are cleaved after reaction.

Water-soluble fluorescent dyes are actively applied to the field ofbiology. In order to incorporate water-soluble fluorescent dyes intobiomolecules, the water-soluble fluorescent dyes should not causephoto-bleaching and quenching under aqueous or hydrophilic solutionconditions, have a high molecular extinction coefficient sufficient toabsorb a great deal of light, be within 500 nm or higher of visible ornear infrared rays far from the fluorescent range of biomolecules and bestable under various pH conditions. However, structures of dyes usefulfor labeling biomolecules are limited due to various conditions.

All dyes are not fluorescent. Researchers in a variety of fields havedeveloped dyes having fluorescent chromophores. Representative examplesof fluorophores known to date include anthranilate, 1-alkylthicisoindoles, pyrrolinones, bimanes, benzoxazole, benzimidazole,benzofuran, naphthalenes, coumarins, stilbenes, carbazoles,phenanthridine, anthracenes, acridines, fluoresceins, eosins,rhodamines, pyrenes, chrysenes and the like. Derivatives similar tothese fluorophores are also researched. These fluorophores areincorporated into various reactive groups to be bound to biomoleculesand are thus commercially available as various products.

It is noted that these various fluorescent dyes should exhibit strongfluorescence in a medium in which most biomolecules are present, thatis, an aqueous solution, in order that the dyes exhibit fluorescenceapplicable to the field of biology. The most commonly used fluorescentdyes for such application are xanthene-based fluorescein and rhodamine,and polymethine-based cyanine.

Cyanine dyes were first applied to biomolecular-labeling by Dr.Waggoner's team in the Carnegie Mellon University near the end of the1980's. Dr. Waggoner's team found that binding of cyanine dyes mainlyused for cloth dying or optical recording media, into which reactivegroups that can be linked to proteins are incorporated, to proteins,causes expression of fluorescence and then reported the followingseveral articles.

-   [Document 1] Ernst, L. A., Gupta, R. K., Mujumdar, R. B., and    Waggoner, A. S. (1989) Cyanine Dye Labeling Reagents for Sulfhydryl    groups. Cytometry 10, 3-10.-   [Document 2] Mujumdar, R. B., Ernst, L. A., Mujumdar, S. R., and    Waggoner, A. S. (1989) Cyanine Dye Labeling Reagents containing    Isothiocyanate groups. Cytometry 10, 11-19.-   [Document 3] Southwick, P. L., Ernst, L. A., Tauriello, E. W.,    Stephen, R. P., Mujumdar, R. B., Mujumdar, S. R., Clever, H. A., and    Waggoner, A. S. (1990) Cyanine Dye Labeling    reagents—Carboxymethylindocyanine Succinimidyl Esters. Cytometry 11,    418-430.-   [Document 4] Mujumdar, R. B., Ernst, L. A., Mujumdar, S. R.,    Lewis, C. J., and Waggoner, A. S. (1993) Cyanine Dye Labeling    Reagents: Sulfoindocyanine Succinimidyl Esters. Bioconjugate Chem.    4, 105-111.-   [Document 5] Mujumdar, S. R., Mujumdar, R. B., Grant, C. M., and    Waggoner, A. S. (1996) Cyanine-Labeling Reagents:    Sulfobenzindocyanine Succinimidyl Esters. Bioconjugate Chem. 7,    356-36.

Then, for various applications, numerous researchers introduced proteinnucleophiles, that is, amine, thiol and hydroxyl groups, and pigmentsand fluorescent dyes for labeling biomolecules, into which reactivegroups bound to electrophiles, that is, aldehyde, ketone and carboxylicacid groups, are incorporated.

Generally, cyanine dyes exhibit optical and pH stability, have narrowabsorption and emission wavelength ranges and are fluorescent in therange of 500 to 800 nm. This fluorescence range of cyanine dyes does notoverlap with the self-fluorescence range of biomolecules, thusadvantageously making it easy to analyze. In addition, cyanine dyesexhibit high molecular extinction coefficients, although there areslight differences therebetween depending on characteristics of solventand solubility. The following Formulas represent a generic structure ofcyanine dyes shown in the documents and basic structures of heterocompounds known as derivatives.

Most commercially available cyanine dyes have indole structures ashetero rings and succinimidyl ester as reactive groups. The Formularepresented below is a representative structure of cyanine dyes, whichare commercially available under the trade names Cy3, Cy5 and Cy7 fromGE healthcare Co., Ltd.

Unlike cloth dyes requiring various colors, fluorescent dyes forlabeling biomolecules having a wide fluorescence wavelength range arenot necessarily preferable. This is the reason that wavelengths ofequipment using or measuring fluorescence are limited. Unless novelfluorescence analysis methods or apparatuses are developed, opticalequipment is improved, or performance thereof is suited for dyes,variations in chromogens or structures varying light-absorption orlight-emission wavelength ranges are not significant in view ofcommercialization in the field of dyes for labeling biomolecules.

It is known that like dyes having different chromogens, cyanine dyeshave polymethine as a chromogen, regardless of incorporation of reactivegroups, and thus substantially maintain fluorescence properties andundergo almost no variation in light-absorption and light-emissionwavelengths.

Cyanine-labeling dyes having succinimidyl ester, used for labelingbiomolecules, are dyed in a carbonate or phosphate buffer solution.Generally, the buffer is used in a concentration of 0.1M and thereaction is carried out at room temperature.

A dye is dissolved in N,N-dimethylformamide (DMF) or N,N-dimethylsulfoxide (DMSO). 1 mg of the dye is dissolved in 100 μl of solvent andthe resulting solution is then aliquoted. The dye is used in an excessof 5- to 100-fold equivalents with respect to biomolecules to be stainedbecause, although a protein molecule is used in an amount of oneequivalent, the number of amino, hydroxyl or thiol groups targeted bythe reaction is much greater. Dyes require higher aqueous solutionstability, in order for the dyes to be permeated into complicatedprotein structures and thus react therewith. However, it isdisadvantageous that succinimidyl ester cannot be stably maintained fora long period of time.

SUMMARY OF THE INVENTION

Therefore, the present invention has been made in view of the aboveproblems, and it is one object of the present invention to provide anovel cyanine compound which may be widely used to identify biomoleculessuch as proteins, lipids or carbohydrates in the field of proteomics andoptical molecular imaging, and a method for preparing the same.

In accordance with the present invention, the above and other objectscan be accomplished by the provision of a novel cyanine compoundrepresented by Formula 1 below:

wherein

R₁ and R_(1′) are each independently hydrogen, a sulfonic acid group ora sulfonic acid base;

R₂, R_(2′), R₃ and R_(3′) are each independently hydrogen or a C₁-C₆alkyl group;

R₄ is hydrogen, a C₁-C₆ alkyl group, a carboxyl group,—CONH(CH₂)_(L)SO₂CH═CH₂, —CONH-para-(C₆H₄)SO₂CH═CH₂ or—CONH-meta-(C₆H₄)SO₂CH═CH₂;

B is (CH₂)_(l), para-(C₆H₄) or meta-(C₆H₄);

m and m′ are each independently an integer of 1 to 5; and

L, l and n are each independently an integer of 1 to 5, and a method forpreparing the same.

In accordance with another aspect, provided is a method for labelingbiomolecules, nanoparticles or organic compounds with the compound ofFormula 1 in the presence of various solvents, for example, in a buffersolution (so-called, binding protocol).

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and other advantages of thepresent invention will be more clearly understood from the followingdetailed description taken in conjunction with the accompanyingdrawings, in which:

FIG. 1 illustrates absorbance (at 550 nm) of the compound 1-16 confirmedin Example 22;

FIG. 2A is an image wherein proteins, which were labeled in Example 23and subjected to development by gel electrophoresis, were observed bythe naked eye, and FIG. 2B is a fluorescence image of the proteins;

FIG. 3A is an image wherein proteins, which were labeled in Example 24and subjected to development by gel electrophoresis, were observed bythe naked eye, and FIG. 3B is a fluorescence image of the proteins;

FIG. 4A is an image wherein proteins, which were labeled in Example 25and subjected to development by gel electrophoresis, were observed bythe naked eye, and FIG. 4B is a fluorescence image of the proteins;

FIG. 5A illustrates absorbance (at 650 nm) of the reaction productobtained by reaction of benzyl amine with the GE Cy5 dye and thecompound 1-2 in Comparative Test (1), FIG. 5B shows absorbance (at 650nm) of a reaction product of benzyl alcohol therewith, and FIG. 5C showsabsorbance (650 nm) of the reaction product of benzyl mercaptantherewith;

FIG. 6A is an image wherein proteins, which were labeled in ComparativeTest (2) and subjected to development by gel electrophoresis, wereobserved by the naked eye, and FIG. 6B is a fluorescence image of theproteins;

FIG. 7A illustrates fluorescence intensity of myosin labeled inComparative Test (2) and developed by gel electrophoresis, FIG. 7Billustrates fluorescence intensity of β-galactosidase, FIG. 7Cillustrates fluorescence intensity of phosphorylase B, FIG. 7Dillustrates fluorescence intensity of serum albumin, FIG. 7E illustratesfluorescence intensity of ovalbumin, FIG. 7F illustrates fluorescenceintensity of carbonic anhydrase, FIG. 7G illustrates fluorescenceintensity of trypsin inhibitor, FIG. 7H illustrates fluorescenceintensity of lysozyme, and FIG. 7I illustrates fluorescence intensity ofaprotinin; and

FIG. 8A is an image wherein proteins, which were labeled in ComparativeTest (3) and subjected to development by gel electrophoresis, wereobserved by the naked eye, and FIG. 8B is a fluorescence image of theproteins.

DETAILED DESCRIPTION OF THE INVENTION

The most commonly used dye reactive group for protein staining issuccinimidyl ester that can be bound to the amines of amino acids. Forexample, U.S. Pat. Nos. 5,268,486, 6,043,025 and 6,127,134 (GEhealthcare) and PCT laid-open No 96/33406 illustrate a method forstaining biomolecules such as antibodies or peptides with variouscyanine dye structures into which succinimidyl ester is incorporated.However, this succinimidyl ester is not stable in an aqueous solutionand necessarily produces N-hydroxy succinimide as a by-product, duringstaining.

Accordingly, the present invention aims to develop dye products that donot produce by-products after dying with biomolecules. In addition,taking into consideration the fact that bio-labeling methods usingconventional succinimidyl ester are well known, a dying method enablingeasy labeling of proteins using a conventional method has been developedto exert superior performance. Also, dying methods performed in thepresence of various buffer solutions are suggested.

The cyanine compound of Formula 1 is characterized in that vinyl sulfoneis incorporated therein. Vinyl groups of the vinyl sulfone are bound tonucleophiles of biomolecules in accordance with the following mechanismand the compound of Formula 1 thus does not produce by-products afterreaction with biomolecules.

The cyanine dye compounds of the present invention are designed totarget water as a medium and be stable against heat, when they areapplied to most biomolecules.

In addition, the present invention is directed to a method for preparingthe novel cyanine compound represented by Formula 1.

Hereinafter, the method for preparing the novel cyanine compoundrepresented by Formula 1 will be described in detail.

In accordance with the method for preparing the compound of Formula 1, acompound of Formula 2 reacts with a compound of Formula 3, as depictedin Reaction Scheme 1a, to obtain a compound of Formula 4a, which is usedas a starting material.

In Formulae 2 to 4 and Reaction Scheme 1a, R₁, R₂ and R₃ are defined asin Formula 1. When R₁ of Formula 4a is a sulfonic acid group, thecompound of Formula 4a is treated with an inorganic base represented bygeneral formula “MOH”, preferably, potassium hydroxide or sodiumhydroxide, most preferably, potassium hydroxide, to obtain a compound ofFormula 4b, in which R₁ is a sulfonic acid base, which may be used as astarting material, as depicted in Reaction Scheme 1b below:

In Reaction Scheme 1b, M is potassium or sodium, R₂ and R₃ are definedas in Formula 1, R₁ of Formula 4a is a sulfonic acid group, and R₁ ofFormula 4b is a sulfonic acid base.

In order to prepare the compound of Formula 1, first, the compound ofFormula 4a or 4b reacts with compounds of the following Formulae 5 and7, to obtain a compound of Formula 6a or 6b, as depicted in ReactionSchemes 2 and 3.

In Formulae 5 to 7 and Reaction Schemes 2 and 3, R₁, R₂, R₃, R₄ and mare each independently defined as in Formula 1 and X is a halogen atomselected from the group consisting of fluorine, chlorine, bromine andiodine.

Reaction Scheme 2 illustrates a method for preparing a compound ofFormula 6a wherein R₄ is hydrogen or an alkyl group having 1 to 6 carbonatoms, and Reaction Scheme 3 illustrates a method for preparing acompound of Formula 6b wherein R₄ is a carboxyl group.

Then, the compound of Formula 6a or 6b reacts with the followingcompound of Formula 8 to obtain a compound of Formula 9.

In Formulae 8 and 9, and Reaction Scheme 4, R₁, R₂, R₃, R₄, m and n areeach independently defined as in Formula 1, and A is hydrogen or anacetyl group.

The compound of Formula 8 is N,N-diphenyl formamidine (DPF), when n is1, the compound of Formula 8 is malondialdehyde dianil hydrochloride(MDH), when n is 2, and the compound of Formula 8 is glutaconaldehydedianil hydrochloride (GDH), when n is 3.

Then, the compound of Formula 9 reacts with a compound of Formula 6b toobtain a compound of Formula 10, as depicted in Reaction Scheme 5 below.

In Formula 10 and Reaction Scheme 5, R₁, R₂, R₃, R₄, m and n are eachindependently defined as in Formula 1. The two R₁, may be the same as ordifferent from each other, the two R₂ may also be the same as ordifferent from each other, and the two R₃ may also be the same as ordifferent from each other. In addition, the two m may also be the sameas or different from each other.

Then, the compound of Formula 10 reacts with, 1′-carbonyl diimidazole(CDI) or N,N-disuccinimidyl carbonate (DSC) to obtain a compound ofFormula 11a or 11b, as depicted in Reaction Scheme 6 below.

In Formulae 11a and 11b, R₁, R₂, R₃, R₄, m and n are each independentlydefined as in Formula 1. The two R₁ may be the same as or different fromeach other, the two R₂ may also be the same as or different from eachother, and the two R₃ may also be the same as or different from eachother. In addition, the two m may also be the same as or different fromeach other.

In Reaction Scheme 6, R₁, R₂, R₃, R₄, m and n are each independentlydefined as in Formula 1, R₆ is an imidazole group in Formula 11a, and R₆is a succinimidyloxy group in Formula 11b. The two R₁ may be the same asor different from each other, the two R₂ may also be the same as ordifferent from each other, and the two R₃ may also be the same as ordifferent from each other. In addition, the two m may be also the sameas or different from each other.

Then, the compound of Formula 11a or 11b reacts with a compoundrepresented by Formula 12 in the presence of a Hunig's base to obtain acompound of Formula 1, as depicted in Reaction Scheme 7 below.

In Formula 12 and Reaction Scheme 7, R₁, R₂, R₃, R₄, m and n are eachindependently defined as in Formula 1, R₅ is a halogen atom selectedfrom the group consisting of fluorine, chlorine, bromine and iodine, ora sulfato group (—OSO₃H), B is (CH₂)₁, p-(C₆H₄) or m-(C₆H₄), and 1 is aninteger of 1 to 5. The two R₁ may be the same as or different from eachother, the two R₂ may also be the same as or different from each other,and the two R₃ may also be the same as or different from each other. Inaddition, the two m may also be the same as or different from eachother.

In addition, the present invention is directed to a method for labelingbiomolecules, nanoparticles or organic compounds containing an aminegroup, a hydroxyl group or a thiol group using the novel cyaninecompound of Formula 1 (binding protocol).

Preferably, the biomolecules are selected from the group consisting ofproteins, peptides, carbohydrates, sugars, lipids, antibodies,proteoglycans, glycoproteins and siRNA.

The labeling is carried out by binding the compound of Formula 1 tobiomolecules, nanoparticles or organic compounds, specifically, to theamine, hydroxyl or thiol groups present in the biomolecules,nanoparticles or organic compounds through reaction of vinyl sulfonepresent in the compound of Formula 1 with the amine, hydroxyl or thiolgroup.

Like conventional cyanine dyes having succinimidyl ester, the compoundof Formula 1 can readily stain proteins through reaction therewith.

Accordingly, the labeling is carried out by reacting the compound ofFormula 1 with the biomolecules, nanoparticles or organic compounds atpH 5 to 12 using, as a solvent, a buffer solution selected from thegroup consisting of a phosphate buffer solution, a carbonate buffersolution and a tris buffer solution, an organic solvent selected fromthe group consisting of dimethyl sulfoxide, dimethyl formamide,methanol, ethanol and acetonitrile, or water. The reaction is carriedout at a temperature of 20 to 80° C. for 30 minutes to 48 hours.

Also, the present invention is directed to a material selected frombiomolecules, nanoparticles and organic compounds labeled with thecompound of Formula 1.

Biomolecules are generally dissolved in a predetermined buffer solutionin a package unit. In order to secure stability of biomolecules,additional buffer solutions or pH are frequently required, makingstability control through parameters difficult. The compound of Formula1 readily reacts with proteins under various buffer conditions, reactiontemperatures and pH to express fluorescence, thus being suitable for usein biomolecular labeling.

EXAMPLES

Hereinafter, the present invention will be described with reference toExamples and Comparative tests in detail. The following examples are forillustrative purposes only and are not intended to limit the scope ofthe present invention.

First, test apparatuses, analysis equipment and reagents used forExamples and Comparative tests will be described in detail.

The apparatuses used herein for FT-NMR spectroscopic analysis wereAvance 300 and 400 (Bruker Co. Ltd.) and 1200L Quadrupole was used forLC/MS (Varian Co. Ltd.) in accordance with electrospray ionization(ESI). Voyager MALDI-TOF DE was used as a mass spectrometer forMALDI-TOF M/S.

The absorption wavelengths and maximum absorption wavelengths ofsynthesized dyes were measured with an HP 8452 diode arrayspectrophotometer (Hewlett-Packard), and luminescence values atluminescent wavelengths and maximum luminescent wavelengths wereobtained using LS-55 (Perkin Elmer Co.).

Column chromatography to separate and purify organic compounds wascarried out using kiesel gel 60 (230-400 mesh, Merck& Company Inc.) as asilica gel in the case of a normal phase. A glass substrate on whichsilica gel 60 GF254 (0.25 mm, Merck) was coated was used for thin layerchromatography (TLC). The identification of the compound through TLC wascarried out using ultraviolet light at 254 nm and 365 nm, or a 20 to 30%ethanolic phosphomolybdic acid (PMA) solution or KMnO₄ as a chromogen.In the case of reverse phase, TLC was carried out using a glasssubstrate on which silica gel 60 RP-18 F_(254S) (0.25 mm, Merck) wascoated, and column chromatography was carried out using a LichroprepRP-18 reverse phase column (40 to 63 μm, Merck) coupled to FractionCollector R-660 as an apparatus for medium pressure liquidchromatography (MPLC, Buchi). HPLC was carried out using Bondapak C18 10μm 125A (Waters) coupled to 1100 series (Agilent).

The apparatus for gel electrophoresis used herein was PowerPac BasicPower Supply (Catalog No. 164-5050, BIO-RAD) coupled to an SE 260mini-vertical gel electrophoresis unit (Amersham Biosciences). PAGErGold Precast Gels (Polyacrylamide gels for protein electrophoresis, 10to 20% Tris-Glycine gels, Catalog No. 59506, Lonza) were used as gels.The running and loading buffers for SDS-PAGE tests were directlyprepared under the following conditions prior to use.

Preparation of 5× Running Buffer

3 g of Tris (Trizma base, Sigma)

14.4 g of Glycine (Sigma)

100 mL of distilled water refrigerated after preparation

Preparation of 5× Loading Buffer

0.6 mL of 1 M Tris (Trizma base, Sigma)

5 mL of 50% glycerol

2 mL of 10% SDS

0.5 mL of 2-mercaptanethanol

1.9 mL of 10% distilled water (instead of bromophenol blue)

Proteins used herein were Size Markers commercially available from GEHealthcare Co., Ltd. and Takara Co., Ltd. A Geliance 600 (Perkin Elmer)was used to observe the labeled biomolecules and measure fluorescenceintensity. Light sources used herein were Geliance UV Epi (Catalog No.L7110026) and Geliance Blue Epi (Catalog No. L7110027). The measurementwas carried out using a UV Filter, Geliance Short Pass Filter (500-600nm), Geliance Long Pass Filter (580 to 660 nm), and Geliance Blue LightFilter (550-600 nm) as filters in accordance with fluorescencewavelengths.

Reagents used herein were products available from Aldrich Co., Ltd. andTCI Co., Ltd. Solvents requiring purification were purified inaccordance with a known method prior to use. Unless specificallymentioned, all reactions were carried out under nitrogen current. NMRsolvents used herein were DMSO-d₆ or D₂O available from Aldrich Co.,Ltd. and Cambridge Isotope Laboratories Inc. Relative positions ofsignals were determined based on tetramethylsilane (TMS) in a solvent oran NMR solvent. Chemical shift was expressed in ppm from a standardmaterial and data of chemical shift multiplicity (s=singlet, d=doublet,t=triplet, m=multiplet), intergration and coupling constant (Hz) weresequentially recorded.

Example 1 Preparation of compound 1-1 (1) Synthesis of Compound 4-1

p-hydrazinobenzenesulfonic acid (10 g, 53 mmol, 1 eq, Aldrich) and3-methyl-2-butanone (17.18 mL, 160 mmol, 3.02 eq, TCI) were added toacetic acid (30 mL), and the resulting mixture was heated under refluxfor 4 hours. The reaction mixture was allowed to cool to ambienttemperature and the resulting solid particles were filtered. Thefiltrate was washed with ethyl acetate two or three times and driedunder reduced pressure (11.34 g, 89%).

R_(f)=0.68 (RP-C18, acetonitrile/water 1:4 v/v)

A solution of the solid substance (5.073 g, 21.2 mmol, 1 eq) thusprepared in methanol (35 mL) was added dropwise to a solution ofpotassium hydroxide (1.427 g, 25.4 mmol, 1.2 eq) in propanol (35 mL),and the mixture was stirred at ambient temperature for 24 hours andfiltered to obtain a yellow particulate solid (5.35 g, 90%).

R_(f)=0.68 (RP-C18, acetonitrile/water 1:4 v/v)

¹H NMR (300 MHz, D₂O): δ 7.60 (s, 1H), 7.58 (d, 1H, J=8.32 Hz), 7.32 (d,1H, J=7.99 Hz), 2.08 (s, 3H), 1.06 (s, 6H)

(2) Synthesis of Compound 6a-1

The compound 4-1 (20 g, 72.1 mmol, 1 eq) and ethyl iodide (110 mL, 1.375mmol, 19 eq, TCI) were added to the solid thus obtained and the mixturewas heated under reflux for 24 hours. The reaction mixture was allowedto cool to ambient temperature, the ethyl iodide was removed, and theresidue was washed with 50 mL of acetone three or four times, filtered,and dried under reduced pressure at 40° C. to obtain a pink solid (18.37g, 95%).

R_(f)=0.18 (RP-C18, acetonitrile/water 1:4 v/v)

¹H NMR (400 MHz, D₂O): δ 7.99 (s, 1H), 7.88 (d, 1H, J=8.23 Hz), 7.80 (d,1H, J=8.46 Hz), 4.43 (m, 2H), 1.52-1.40 (m, 12H)

LC/MS, C₁₃H₁₈NO₃S⁺, calculated value: 268.1, measured value: 268.16

(3) Synthesis of Compound 6B-1

The compound 4-1 (2.774 g, 10 mmol, 1 eq) and 6-bromo-n-hexanoic acid(2.34 g, 12 mmol, 1.2 eq, Aldrich) were heated under reflux in 15 mL of1,2-dichlorobenzene for 12 hours. The reaction mixture was allowed tocool to ambient temperature, the solvent was removed, isopropyl alcoholwas added to the residue, and the resulting mixture was filtered anddried under reduced pressure to obtain a pink solid (2.653 g, 75%).

R_(f)=0.08 (RP-C18, acetonitrile/water 1:4 v/v)

¹H NMR (400 MHz, D₂O): δ 8.00 (s, 1H), 7.90 (d, 1H, J=8.86 Hz), 7.77 (d,1H, J=8.43 Hz), 4.37 (t, 2H, J=7.46 Hz), 2.25 (t, 2H, J=7.01 Hz), 1.85(m, 2H), 1.57-1.26 (m, 13H)

LC/MS, C₁₇H₂₄NO₅S⁺, calculated value: 354.14, measured value: 354.18

(4) Synthesis of Compound 9-1

The compound 6a-1 (16 g, 59.8 mmol, 1 eq) and DPF (13.2 g, 67.3 mmol,1.125 eq, TCI) were added to a solution consisting of 40 mL of aceticacid and 40 mL of anhydrous acetic acid, and the resulting mixture washeated under reflux for 4 hours. The reaction mixture was allowed tocool to ambient temperature, the solution was removed, ethyl acetate wasadded to the residue to produce a solid, and the resulting solid wasfiltered and dried under reduced pressure (12.97 g, 57%).

R_(f)=0.25 (RP-C18, acetonitrile/water 1:4 v/v)

¹H NMR (300 MHz, DMSO-d₆): δ 7.85 (s, 1H), 7.70 (dd, 1H, J=1.35 Hz, 1.32Hz), 7.53-7.45 (m, 7H), 7.29 (dd, 1H, J=1.92 Hz, 6.66 Hz), 4.13 (m, 2H),1.70 (s, 6H), 1.32 (t, 3H, J=7.05 Hz)

LC/MS, C₂₀H₂₃N₂O₃S⁺, calculated value: 371.14, measured value: 370.98

(5) Synthesis of Compound 10-1

The compound 9-1 (1.01 g, 2.429 mmol, 1 eq) and the compound 6b-1 (0.86g, 2.429 mmol, 1 eq) were added to a solution consisting of 5 mL ofanhydrous acetic acid and 5 mL of pyridine and the mixture was allowedto react at 110° C. for 4 hours. The reaction mixture was allowed tocool to ambient temperature, and a solid was precipitated throughaddition of ethyl acetate, was filtered and dried under reducedpressure. The resulting product was purified by RP-C18 reverse phasechromatography using 15% acetonitrile aqueous solution as an eluent toobtain a pure compound 10-1 (0.37 g, 24%).

R_(f)=0.70 (RP-C18, acetonitrile/water 3:7 v/v)

¹H NMR (300 MHz, D₂O): δ 8.38 (t, 1H, J=13.5 Hz), 7.78 (s, 2H), 7.73 (t,2H, J=7.42 Hz), 7.23 (dd, 2H, J=5.25 Hz, 7.97 Hz), 6.24 (dd, 2H, J=4.79Hz, 4.56 Hz), 3.97 (m, 4H), 2.23 (t, 2H, J=7.26 Hz), 1.73-1.20 (m, 21H)

LC/MS, C₃₁H₃₉N₂O₈S₂ ⁺, calculated value: 631.21, measured value: 631.31

λ_(abs) (water): 549 nm, λ_(fl) (water): 573 nm

(6) Synthesis of Compound 1-1

The compound 10-1 (110 mg, 0.174 mmol, 1eq) was dissolved in DMF (22 mL)and the temperature was elevated to 55° C. 0.11 mL of pyridine was addedto the solution and a solution of DSC (135 mg, 0.527 mmol, 3.02 eq,Aldrich) in DMF (2.5 mL) was added dropwise thereto. The resultingmixture was stirred for one hour, a red solid was a precipitated throughaddition of ethyl acetate, was washed several times with ethyl acetateand ether and filtered. The filtrate was dissolved in DMF (20 mL), 0.302mL of Hunig's base was added thereto, a solution of2-(2′-chloroethylsulfonyl)ethylamine hydrochloride (36 mg, 0.174 mmol, 1eq) dissolved in 1 mL of DMF was dropwise added thereto, and theresulting mixture was stirred for 12 hours or longer. The reactionmixture was extracted in water and methylene chloride (dichloromethane),and distilled under reduced pressured at 35 to 40° C. to remove thesolvent. The residue was purified by RP-C18 reverse phase chromatographyusing 15% acetonitrile aqueous solution as an eluent to obtain a purecompound 1-1 (106.4 mg, 82%).

R_(f)=0.65 (RP-C18, acetonitrile/water 3:7 v/v)

¹H NMR (400 MHz, DMSO-d₆): δ 8.32 (t, 1H, J=13.4 Hz), 8.02 (t, 1H, J=4.9Hz), 7.80 (s, 2H), 7.67 (m, 2H), 7.39 (dd, 2H, J=2.88 Hz, 3 Hz), 6.96(dd, 1H, J=9.92 Hz, 9.92 Hz), 6.53 (dd, 2H, J=4.24 Hz, 4.32 Hz), 6.23(m, 2H), 4.17-4.09 (m, 4H), 3.21 (m, 2H), 2.04 (t, 2H, J=6.96 Hz),1.69-1.28 (m, 23H)

LC/MS, C₃₅H₄₄N₃O₉S₃ ⁻, calculated value: 746.22, measured value: 746.27

λ_(abs) (water): 549 nm (ε=1.222×10⁵M⁻¹ cm⁻¹), λ_(fl) (water): 574 nm

Example 2 Preparation of Compound 1-2 (1) Synthesis of Compound 9-2

The compound 6a-1 (2.2 g, 8.23 mmol, 1 eq) and MDH (2.55 g, 9.88 mmol,1.2 eq, TCI) were heated under reflux in a solution consisting of 10 mLof acetic acid and 10 mL of anhydrous acetic acid for 4 hours. Thereaction mixture was allowed to cool to ambient temperature, the solventwas removed, a solid is precipitated through addition of ethyl acetatewas filtered and then dried under reduced pressure (3.47 g, 96%).

R_(f)=0.20 (RP-C18, acetonitrile/water 1:4 v/v)

(2) Synthesis of Compound 10-2

The compound 9-2 (6.40 g, 14.6 mmol, 1 eq) and the compound 6b-1 (5.12g, 14.6 mmol, 1 eq) were added to 80 mL of pyridine and the resultingmixture was allowed to react at 60° C. for 4 hours. The reaction mixturewas allowed to cool to ambient temperature, and a blue solid wasprecipitated through addition of ethyl acetate, was filtered and driedunder reduced pressure. The resulting product was purified by RP-C18reverse phase chromatography using 25% acetonitrile aqueous solution asan eluent to obtain a pure compound 10-2 (2.09 g, 22%).

R_(f)=0.58 (RP-C18, acetonitrile/water 3:7 v/v)

¹H NMR (400 MHz, DMSO-d₆): δ 8.34 (t, 2H, J=13.2 Hz), 7.80 (s, 2H), 7.63(d, 2H, J=8.16 Hz), 7.30 (dd, 2H, J=2.80 Hz, 2.76 Hz), 6.58 (t, 1H,J=12.2 Hz), 6.30 (dd, 2H, J=8.64 Hz, 8.56 Hz), 4.13-4.06 (m, 4H), 1.98(t, 2H, J=6.84 Hz), 1.72-1.18 (m, 21H)

LC/MS, C₃₃H₃₉N₂O₈S₂ ⁻, calculated value: 655.22, measured value: 655.24

λ_(abs) (water): 647 nm, λ_(fl) (water): 678 nm

(3) Synthesis of Compound 1-2

The compound 10-2 (20 mg, 0.0305 mmol, 1 eq) was dissolved in DMF (4 mL)and the temperature was elevated to 55° C. 0.02 mL of pyridine was addedto the solution and a solution of DSC (40 mg, 0.156 mmol, 5.13 eq) inDMF (0.35 mL) was dropwise added thereto. The resulting mixture wasstirred for one hour and a blue solid precipitated through addition ofethyl acetate was filtered and washed several times with ethyl acetateand ether. The filtrate was dissolved in DMF (4 mL), 40 mg of Hunig'sbase was added thereto, a solution of 2-(2′-chloroethylsulfonyl)ethylamine hydrochloride (6.5 mg, 0.0312 mmol, 1.03 eq) dissolved in 0.1 mLof DMF was dropwise added thereto, and the resulting mixture was stirredat ambient temperature for 12 hours or longer. The reaction mixture wasextracted in water and methylene chloride (dichloromethane), anddistilled under reduced pressured at 35 to 40° C. to remove the solvent.The residue was purified by RP-C18 reverse phase chromatography using a15% acetonitrile aqueous solution as an eluent to obtain a pure compound1-2 (18.5 mg, 79%).

R_(f)=0.58 (RP-C18, acetonitrile/water 3:7 v/v)

¹H NMR (300 MHz, DMSO-d₆): δ 8.35 (t, 2H, J=13.0 Hz), 8.00 (t, 1H,J=5.21 Hz), 7.81 (s, 2H), 7.62 (d, 2H, J=10.3 Hz), 7.31 (d, 2H, J=7.50Hz), 6.98 (dd, 1H, J=9.91 Hz, 9.92 Hz), 6.62 (t, 1H, J=12.3 Hz),6.32-6.21 (m, 4H), 4.13-4.07 (m, 4H), 3.25 (m, 2H), 2.03 (t, 2H, J=7.13Hz), 1.68-1.23 (m, 23H)

LC/MS, C₃₇H₄₈N₃O₉S₃ ⁺, calculated value: 774.25, measured value: 774.29

λ_(abs) (water): 648 nm (c=2.012×10⁵M⁻¹cm⁻¹), λ_(fl) (water): 679 nm

Example 3 Preparation of Compound 1-3 (1) Synthesis of Compound 9-3

The compound 6a-1 (2.2 g, 8.23 mmol, 1 eq) and GDH (2.812 g, 9.87 mmol,1.2 eq, TCI) were added to 10 mL of anhydrous acetic acid and theresulting mixture was allowed to react at 100° C. for one hour. Thereaction mixture was allowed to cool to ambient temperature and a solidprecipitated using ethyl acetate was filtered and dried under reducedpressure (2.92 g, 76%).

R_(f)=0.80 (RP-C18, acetonitrile/water 1:2 v/v)

(2) Synthesis of Compound 10-3

The compound 9-3 (7.2 g, 17.5 mmol, 1 eq) and the compound 6b-1 (5.94 g,17.5 mmol, 1 eq) were dissolved in 108 mL of pyridine and the resultingmixture was allowed to react at 40° C. for one hour. The reactionmixture was allowed to cool to ambient temperature, and a green solidwas precipitated through addition of ethyl acetate was filtered anddried under reduced pressure. The residue was purified by RP-C18 reversephase chromatography using 30% acetonitrile aqueous solution as aneluent to obtain a pure compound 10-3 (2.59 g, 23%).

R_(f)=0.38 (RP-C18, acetonitrile/water 3:7 v/v)

¹H NMR (400 MHz, DMSO-d₆): δ 7.87 (t, 2H, J=12.8 Hz), 7.72 (m, 3H), 7.50(d, 2H, J=11.9 Hz), 7.28 (d, 2H, J=8.24 Hz), 6.58-6.49 (m, 2H), 6.36 (d,2H, J=13.7 Hz), 4.10-4.03 (m, 4H), 1.98 (t, 2H), 1.70-1.22 (m, 21H)

LC/MS, C₃₅H₄₁N₂O₈S₂ ⁻, calculated value: 681.23, measured value: 681.28

λ_(abs) (water): 748 nm, λ^(fl) (water): 786 nm

(3) Synthesis of Compound 1-3

The compound 10-3 (20.8 mg, 0.0305 mmol, 1 eq) was dissolved in DMF (4mL) and the temperature was elevated to 55° C. 0.02 mL of pyridine wasadded to the solution and a solution of DSC (23.6 mg, 0.0921 mmol, 3.02eq) in DMF (0.3 mL) was dropwise added thereto. The resulting mixturewas stirred for one hour, a green solid was precipitated throughaddition of ethyl acetate was filtered, while washing with ethyl acetateand ether several times. The filtrate was dissolved in DMF (4 mL), 40 mgof Hunig's base was added thereto, a solution of2-(T-chloroethylsulfonyl)ethyl amine hydrochloride (6.5 mg, 0.0312 mmol,1.03 eq) dissolved in 0.1 mL of DMF was dropwise added thereto, and theresulting mixture was stirred at ambient temperature for 12 hours orlonger. The reaction mixture was extracted in water and methylenechloride (dichloromethane), and distilled under reduced pressured at 35to 40° C. to remove the solvent. The residue was purified by RP-C18reverse phase chromatography using 15% acetonitrile aqueous solution asan eluent to obtain a pure compound 1-3 (17.9 mg, 73%).

R_(f)=0.50 (RP-C18, acetonitrile/water 3:7 v/v)

¹H NMR (400 MHz, DMSO-d₆): δ 8.03 (t, 1H), 7.88-7.60 (m, 7H), 7.29 (dd,2H, J=3.24 Hz, 3.23 Hz), 6.97 (m, 1H), 6.54 (m, 2H), 6.39-6.22 (m, 4H),4.10-4.03 (m, 4H), 3.23 (m, 2H), 2.03 (t, 2H, J=7.08 Hz), 1.62-1.12 (m,23H)

LC/MS, C₃₉H₅₀N₃O₉S₃ ⁺, calculated value: 800.27, measured value: 800.32

λ_(abs) (water): 748 nm (ε=1.464×10⁵M⁻¹cm⁻¹), λ_(fl) (water): 790 nm

Examples 4 to 15

The compounds of Examples 4 to 15 (compounds 1-4 to 1-15) were preparedusing a method similar to that of Examples 1 to 3. Data of thestructures of these compounds are given below:

Example 4 Preparation of Compounds 1-4 (1) Compound 6a-2

(19.81 g, 98%)

R_(f)=0.45 (RP-C18, acetonitrile/water 1:4 v/v)

(2) Compound 9-4

(10.28 g, 85%)

R_(f)=0.10 (RP-C18, acetonitrile/water 1:4 v/v)

(3) Compound 10-4

(3.06 g, 20.6%)

R_(f)=0.49 (RP-C18, acetonitrile/water 3:7 v/v)

¹H NMR (300 MHz, DMSO-d₆): δ 8.34 (t, 1H, J=13.2 Hz), 7.78 (s, 2H), 7.65(d, 2H, J=8.04 Hz), 7.39 (m, 2H), 6.56 (dd, 2H, J=13.16 Hz, 13.44 Hz),4.10 (m, 4H), 1.88 (t, 2H, J=6.88 Hz), 1.77-1.38 (m, 21H), 0.96 (t, 3H,J=7.24 Hz)

LC/MS, C₃₂H₃₉N₂O₈S₂ ⁻, calculated value: 643.22, measured value: 643.29

λ_(abs) (water): 550 nm, λ_(fl) (water): 574 nm

(4) Compound 1-4

(39.5 mg, 84%)

R_(f)=0.55 (RP-C18, acetonitrile/water 3:7 v/v)

¹H NMR (400 MHz, DMSO-d₆): δ 8.35 (t, 1H, J=13.4 Hz), 7.99 (m, 1H), 7.79(s, 2H), 7.66 (d, 2H, J=7.86 Hz), 7.39 (dd, 2H, J=8.68 Hz, 8.60 Hz),6.96 (dd, 1H, J=9.90 Hz, 9.96 Hz), 6.51 (t, 2H, J=9.25 Hz), 6.23 (m,2H), 4.09 (m, 4H), 3.21 (m, 2H), 2.05 (m, 2H), 1.76-1.24 (m, 23H), 0.96(t, 3H, J=6.99 Hz)

LC/MS, C₃₆H₄₆N₃O₉S₃ ⁻, calculated value: 760.24, measured value: 760.30

*λ_(abs) (water): 551 nm, λ_(fl) (water): 576 nm

Example 5 Preparation of Compounds 1-5 (1) Compound 9-5

(9.12 g, 71%)

R_(f)=0.13 (RP-C18, acetonitrile/water 1:4 v/v)

(2) Compound 10-5

(2.17 g, 16%)

R_(f)=0.49 (RP-C18, acetonitrile/water 3:7 v/v)

¹H NMR (400 MHz, DMSO-d₆): δ 8.34 (t, 2H, J=12.8 Hz), 7.79 (s, 2H), 7.61(d, 2H, J=7.80 Hz), 7.31 (t, 2H, J=9.2 Hz), 6.59 (t, 1H, J=12.1 Hz),6.30 (dd, 2H, J=3.53 Hz, 3.47 Hz), 4.05 (m, 4H), 1.97 (t, 2H, J=6.96Hz), 1.72-1.22 (m, 21H), 0.92 (t, 3H, J=7.24 Hz)

LC/MS, C₃₄H₄₁N₂O₈S₂ ⁻, calculated value: 669.23, measured value: 669.30

λ_(abs) (water): 649 nm, λ_(fl), (water): 668 nm

(3) Compound 1-5

(43.1 mg, 88.3%)

R_(f)=0.55 (RP-C18, acetonitrile/water 3:7 v/v)

¹H NMR (300 MHz, DMSO-d₆): δ 8.34 (t, 2H, J=12.6 Hz), 7.97 (t, 1H), 7.80(s, 2H), 7.62 (d, 2H, J=7.8 Hz), 7.31 (t, 2H), 6.95 (dd, 1H, J=9.88 Hz,9.96 Hz), 6.58 (t, 1H, J=11.9 Hz), 6.33-6.21 (m, 4H), 4.06 (m, 4H), 3.21(t, 2H, J=6.88 Hz), 2.03 (t, 2H, J=6.96 Hz), 1.74-1.24 (m, 23H), 0.93(t, 3H, J=7.24 Hz)

LC/MS, C₃₈H₄₈N₃O₉S₃ ⁻, calculated value: 786.26, measured value: 786.34

λ_(abs) (water): 649 nm, λ_(fl) (water): 672 nm

Example 6 Preparation of Compounds 1-6 (1) Compound 9-6

(5.49 g, 65%)

R_(f)=0.55 (RP-C18, acetonitrile/water 1:2 v/v)

(2) Compound 10-6

(2.60 g, 20%)

R_(f)=0.37 (RP-C18, acetonitrile/water 3:7 v/v)

¹H NMR (300 MHz, DMSO-d₆): δ 7.92-7.73 (m, 5H), 7.62 (d, 2H, J=8.13 Hz),7.30 (t, 2H, J=7.62 Hz), 6.50 (t, 2H), 6.38 (dd, 2H, J=3.84 Hz, 4.05Hz), 4.04 (m, 4H), 2.01 (t, 2H, J=6.96 Hz), 1.75-1.23 (m, 21H), 0.94 (t,3H, J=7.29 Hz)

LC/MS, C₃₆H₄₃N₂O₈S₂ ⁻, calculated value: 695.25, measured value: 695.29

λ_(abs) (water): 749 nm, λ_(fl) (water): 790 nm

(3) Compound 1-6

(35.5 mg, 70%)

R_(f)=0.48 (RP-C18, acetonitrile/water 3:7 v/v)

¹H NMR (400 MHz, DMSO-d₆): δ 7.98 (m, 1H), 7.87 (t, 2H, J=10.6 Hz), 7.73(m, 3H), 7.61 (d, 2H, J=8.08 Hz), 7.29 (dd, 2H, J=8.21 Hz, 8.40 Hz),6.96 (dd, 1H, J=10.1 Hz, 10.1 Hz), 6.54 (t, 2H, J=12.1 Hz), 6.36 (t, 2H,J=14.1 Hz), 6.23 (m, 2H), 4.03 (m, 4H), 3.22 (m, 2H), 2.03 (t, 2H,J=7.26 Hz), 1.74-1.22 (m, 23H), 0.93 (t, 3H, J=7.22 Hz)

LC/MS, C₄₀H₅₀N₃O₉S₃ ⁻, calculated value: 812.27, measured value 812.35

λ_(abs) (water): 749 nm, λ_(fl) (water): 789 nm

Example 7 Preparation of Compounds 1-7 (1) Compound 6a-3

(13.45 g, 82%)

R_(f)=0.13 (RP-C18, acetonitrile/water 1:4 v/v)

(2) Compound 9-7

(6.48 g, 52%)

R_(f)=0.33 (RP-C18, acetonitrile/water 1:4 v/v)

(3) Compound 10-7

(0.94 g, 27%)

R_(f)=0.80 (RP-C18, acetonitrile/water 3:7 v/v)

¹H NMR (300 MHz, D₂O): δ 7.95 (m, 1H), 7.84-7.62 (m, 4H), 7.46 (d, 2H,J=7.89 Hz), 6.51 (d, 2H, J=8.22 Hz), 4.41 (t, 2H, J=8.07 Hz), 3.56 (s,3H), 1.90 (m, 2H), 1.70-1.24 (m, 18H)

MALDI-TOF M/S, C₃₀H₃₇N₂O₈S₂ ⁺, calculated value: 617.2, measured value:617.53

λ_(abs) (water): 546 nm, λ_(fl) (water): 570 nm

(4) Compound 1-7

(37.2 mg, 82%).

R_(f)=0.71 (RP-C18, acetonitrile/water 3:7 v/v)

¹H NMR (300 MHz, DMSO-d₆): δ 8.31 (t, 1H), 8.06 (t, 1H, J=5.86 Hz), 7.64(s, 2H), 7.65 (m, 2H), 7.38 (dd, 2H, J=4.32 Hz, 4.36 Hz), 6.97 (dd, 1H,J=9.36 Hz, 9.96 Hz), 6.46 (dd, 2H, J=5.24 Hz, 5.36 Hz), 6.23 (m, 2H),4.08 (m, 4H), 3.64 (s, 3H), 3.21 (t, 2H, J=6.72 Hz), 2.04 (t, 2H, J=6.84Hz), 1.68-1.23 (m, 20H)

LC/MS, C₃₄H₄₂N₃O₉S₃ ⁻, calculated value: 732.21, measured value: 732.47

λ_(abs) (water): 549 nm, λ_(fl) (water): 568 nm

Example 8 Preparation of Compound 1-8 (1) Compound 9-8

(2.05 g, 74%)

R_(f)=0.25 (RP-C18, acetonitrile/water 1:4 v/v)

(2) Compound 10-8

(0.88 g, 15%)

R_(f)=0.63 (RP-C18, acetonitrile/water 3:7 v/v)

¹H NMR (300 MHz, DMSO-d₆): δ 7.91 (t, 2H, J=13.7 Hz), 7.74-7.69 (m, 4H),7.24 (t, 2H, J=8.43 Hz), 6.45 (t, 1H, J=12.5 Hz), 6.16-6.11 (m, 2H),3.98 (t, 2H, J=5.95 Hz), 3.49 (s, 3H), 2.11 (t, 2H, J=7.06 Hz),1.73-1.34 (m, 18H)

LC/MS, C₃₂H₃₇N₂O₈S₂ ⁻, calculated value: 641.20, measured value: 641.27

λ_(abs) (water): 646 nm, λ_(fl) (water): 666 nm

(3) Compound 1-8

(38.1 mg, 81%)

R_(f)=0.63 (RP-C18, acetonitrile/water 3:7 v/v)

¹H NMR (400 MHz, DMSO-d₆): δ 8.34 (t, 2H, J=13.5 Hz), 8.00-7.94 (m, 3H),7.79 (s, 2H), 7.61 (t, 2H, J=8.04 Hz), 7.30 (dd, 2H, J=3.04 Hz, 3.00Hz), 6.97 (dd, 1H, J=10.5 Hz, 10.2 Hz), 6.57 (t, 1H, J=12.2 Hz),6.31-6.21 (m, 4H), 4.06 (m, 2H), 3.58 (s, 3H), 3.21 (t, 2H, J=6.64 Hz),2.02 (t, 2H, J=7.08 Hz), 1.67-1.24 (m, 20H)

LC/MS, C₃₆H₄₄N₃O₉S₃ ⁻, calculated value: 758.22, measured value: 758.47

λ_(abs) (water): 646 nm, λ_(fl) (water): 674 nm

Example 9 Preparation of Compound 1-9 (1) Compound 9-9

(11.49 g, 80.9%)

R_(f)=0.83 (RP-C18, acetonitrile/water 1:2 v/v)

(2) Compound 10-9

(1.33 g, 16%)

*R_(f)=0.43 (RP-C18, acetonitrile/water 3:7 v/v)

¹H NMR (300 MHz, DMSO-d₆): δ 7.80-7.62 (m, 5H), 7.33 (t, 2H, J=13.2 Hz),7.17 (t, 2H, J=7.32 Hz), 6.34 (t, 2H, 12.2 Hz), 6.06 (dd, 2H, J=5.26 Hz,5.05 Hz), 3.92 (m, 2H), 3.44 (s, 3H), 2.10 (t, 2H, J=7.45 Hz), 1.90-1.20(m, 18H)

LC/MS, C₃₄H₃₉N₂O₈S₂ ⁻, calculated value: 667.22, measured value: 667.33

λ_(abs) (water): 746 nm, λ_(fl) (water): 783 nm

(3) Compound 1-9

(40.6 mg, 84%)

R_(f)=0.57 (RP-C18, acetonitrile/water 3:7 v/v)

¹H NMR (400 MHz, DMSO-d₆): δ 8.00 (m, 1H), 7.88-7.86 (m, 2H), 7.73 (m,3H), 7.61 (t, 2H, J=6.64 Hz), 7.28 (dd, 2H, J=8.12 Hz, 8.00 Hz),7.10-6.90 (m, 1H), 6.53-6.49 (m, 2H); 6.34-6.22 (m, 4H), 4.02 (m, 2H),3.57 (s, 3H), 3.22 (t, 2H), 2.03 (t, 2H), 1.74-1.22 (m, 20H)

LC/MS, C₃₈H₄₆N₃O₉S₃ ⁻, calculated value: 784.24, measured value: 784.47

λ_(abs) (water): 746 nm, λ_(fl) (water): 792 nm

Example 10 Preparation of Compound 1-10 (1) Compound 6a-4

(5.65 g, 96%)

R_(f)=0.58 (RP-C18, acetonitrile/water 1:4 v/v)

(2) Compound 9-10

(1.75 g, 56.1%)

R_(f)=0.08 (RP-C18, acetonitrile/water 1:4 v/v)

(3) Compound 10-10

(1.09 g, 23.3%)

R_(f)=0.61 (RP-C18, acetonitrile/water 3:7 v/v)

¹H NMR (400 MHz, DMSO-d₆): δ 8.34 (t, 1H, J=13.4 Hz), 7.86 (s, 2H), 7.79(d, 2H, J=7.72 Hz), 7.38 (d, 2H, J=6.16 Hz), 6.56 (dd, 2H, J=13.6 Hz,13.5 Hz), 4.11 (m, 4H), 1.92 (t, 2H, J=6.96 Hz), 1.76-1.22 (m, 25H),0.92 (t, 3H, J=7.2 Hz)

L/C M/S, C₃₃H₄₁N₂O₈S₂ ⁻, calculated value: 657.23, measured value:657.28

λ_(abs) (water): 551 nm, λ_(fl) (water): 567 nm

(4) Compound 1-10

(187.4 mg, 81.9%)

R_(f)=0.60 (RP-C18, acetonitrile/water 3:7 v/v)

¹H NMR (400 MHz, DMSO-d₆): δ 8.36 (t, 1H), 8.02 (t, 1H), 7.81 (s, 2H),1.68 (d, 2H), 7.40 (dd, 2H), 6.97 (dd, 1H), 6.53 (dd, 2H), 6.23 (m, 2H),4.12 (m, 4H), 3.22 (t, 2H), 2.05 (t, 2H), 1.75-1.18 (m, 27H), 0.93 (t,3H)

LC/MS, C₃₇H₄₈N₃O₉S₃ ⁻, calculated value: 774.26, measured value: 774.53

λ_(abs) (water): 551 nm (ε=1.738×10⁵ M⁻¹cm⁻¹), λ_(fl) (water): 568 nm

Example 11 Preparation of Compound 1-11 (1) Compound 9-11

(2.92 g, 88%)

R_(f)=0.08 (RP-C18, acetonitrile/water 1:4 v/v)

(2) Compound 10-11

(1.15 g, 23.6%)

R_(f)=0.42 (RP-C18, acetonitrile/water 3:7 v/v)

¹H NMR (400 MHz, DMSO-d₆): δ 8.34 (t, 2H, J=12.0 Hz), 7.80 (s, 2H), 7.62(d, 2H, J=7.20 Hz), 7.32 (m, 2H), 6.59 (t, 1H, J=11.5 Hz), 6.31 (m, 2H),4.08 (m, 4H), 2.04 (t, 2H), 1.77-1.21 (m, 25H), 0.90-0.79 (m, 3H)

LC/MS, C₃₅H₄₃N₂O₈S₂ ⁻, calculated value: 683.25, measured value: 683.33

λ_(abs) (water): 649 nm, λ_(fl) (water): 668 nm

(3) Compound 1-11

(185 mg, 76.9%)

R_(f)=0.51 (RP-C18, acetonitrile/water 3:7 v/v)

¹H NMR (400 MHz, DMSO-d₆): δ 8.35 (t, 2H, J=12.4 Hz), 8.00 (t, 1H), 7.80(s, 2H), 7.61 (d, 2H, J=8.32 Hz), 7.30 (dd, 2H, J=4.00 Hz, 4.20 Hz),6.96 (m, 1H), 6.58 (t, 1H), 6.31-6.21 (m, 4H), 4.07 (m, 4H), 3.22 (t,2H, J=6.60 Hz), 2.03 (t, 2H, J=7.04 Hz), 1.53-1.22 (m, 27H), 0.91 (t,3H, J=7.28 Hz)

LC/MS, C₃₉H₅₀N₃O₉S₃ ⁻, calculated value: 800.27, measured value: 800.36

λ_(abs) (water): 649 nm (ε=2.024×10⁵ M⁻¹cm⁻¹), λ_(fl) (water): 670 nm

Example 12 Preparation of Compound 1-12 (1) Compound 9-12

(2.52 g, 71.8%)R_(f)=0.42 (RP-C18, acetonitrile/water 1:2 v/v)

(2) Compound 10-12

(1.26 g, 25.0%)

R_(f)=0.35 (RP-C18, acetonitrile/water 3:7 v/v)

¹H NMR (400 MHz, DMSO-d₆): δ 7.79-7.73 (m, 5H), 7.61 (d, 2H, J=6.84 Hz),7.29 (dd, 2H, J=3.48 Hz, 3.16 Hz), 6.56 (t, 2H, J=12.1 Hz), 6.36 (d, 2H,J=13.6 Hz), 4.04 (m, 4H), 2.01 (t, 2H), 1.50-1.33 (m, 25H), 0.90 (t, 3H,J=7.16 Hz)

LC/MS, C₃₇H₄₅N₂O₈S₂ ⁻, calculated value: 709.26, measured value: 709.34

λ_(abs) (water): 749 nm, λ_(fl) (water): 778 nm

(3) Compound 1-12

(171.7 mg, 69.1%)

R_(f)=0.44 (RP-C18, acetonitrile/water 3:7 v/v)

¹H NMR (400 MHz, DMSO-d₆): δ 8.02-7.72 (m, 6H), 7.63 (d, 2H), 7.28 (m,2H), 6.98 (dd, 1H), 6.55 (t, 2H), 6.36 (dd, 2H), 6.28-6.22 (m, 2H), 4.05(m, 4H), 3.23 (m, 2H), 2.02 (t, 2H), 1.70-1.21 (m, 23H), 0.92 (t, 3H)

LC/MS, C₄₁H₅₂N₃O₉S₃ ⁻, calculated value: 826.29, measured value: 826.80

λ_(abs) (water): 749 nm (c=1.817×10⁵M⁻¹cm⁻¹), λ_(fl) (water): 779 nm

Example 13 Preparation of Compound 1-13 (1) Compound 6b-2

(10.18 g, 64%)

R_(f)=0.13 (RP-C18, acetonitrile/water 1:4 v/v)

(2) Compound 10-13

(1.47 g, 24%)

R_(f)=0.71 (RP-C18, acetonitrile/water 3:7 v/v)

¹H NMR (300 MHz, DMSO-d₆): δ 8.35 (t, 1H, J=13.5 Hz), 7.79 (s, 2H), 7.68(d, 2H, J=8.22 Hz), 7.41 (m, 2H), 6.57 (dd, 2H, J=5.00 Hz, 4.78 Hz),4.18-4.11 (m, 4H), 2.05 (t, 2H), 1.69-0.83 (m, 19H)

LC/MS, C₃₀H₃₅N₂O₈S₂ ⁻, calculated value: 615.18, measured value: 615.28

λ_(abs) (water): 549 nm, λ^(fl) (water): 572 nm

(3) Compound 1-13

(96.8 mg, 75.8%)

R_(f)=0.70 (RP-C18, acetonitrile/water 3:7 v/v)

¹H NMR (400 MHz, DMSO-d₆): ε 8.34 (t, 1H, J=13.7 Hz), 8.07 (m, 1H), 7.80(s, 2H), 7.67 (d, 2H, J=7.60 Hz), 7.39 (dd, 2H, J=3.80 Hz, 3.92 Hz),6.94 (dd, 1H, J=9.96 Hz, 10.1 Hz), 6.51 (d, 2H, J=13.3 Hz), 6.21 (m,2H), 4.08 (m, 4H), 3.21 (t, 2H, J=6.72 Hz), 2.04 (t, 2H, J=6.84 Hz),1.68-1.23 (m, 20H)

LC/MS, C₃₄H₄₄N₃O₉S₃ ⁺, calculated value: 734.22, measured value: 734.07

λ_(abs) (water): 549 nm, λ_(fl) (water): 566 nm

Example 14 Preparation of Compound 1-14 (1) Compound 10-14

(1.66 g, 25.8%)

R_(f)=0.63 (RP-C18, acetonitrile/water 3:7 v/v) ¹H NMR (300 MHz,DMSO-d₆): δ 7.90 (t, 2H, J=13.1 Hz), 7.74-7.68 (m, 4H), 7.24 (d, 2H,J=8.29 Hz), 6.46 (t, 1H, J=12.8 Hz), 6.17 (dd, 2H, J=10.5 Hz, 10.7 Hz),3.98 (dd, 4H, J=7.05 Hz, 6.86 Hz), 2.16 (t, 2H, J=7.15 Hz), 1.83-1.23(m, 18H)

LC/MS, C₃₂H₃₇N₂O₈S₂ ⁻, calculated value: 641.2, measured value: 641.0

λ_(abs) (water): 647 nm, λ^(fl) (water): 675 nm

(2) Compound 1-14

(16.0 mg, 70%)

R_(f)=0.60 (RP-C18, acetonitrile/water 3:7 v/v)

¹H NMR (400 MHz, DMSO-d₆): δ 8.32 (t, 2H, J=12.9 Hz), 8.04 (t, 1H), 7.80(s, 2H), 7.62 (t, 2H, J=6.84 Hz), 7.30 (dd, 2H, J=3.56 Hz, 3.52 Hz),7.00-6.90 (m, 1H), 6.56 (t, 1H), 6.31-6.19 (m, 4H), 4.10 (m, 4H), 3.20(m, 2H), 2.11 (t, 2H), 1.67-0.95 (m, 21H)

LC/MS, C₃₆H₄₄N₃O₉S₃ ⁻, calculated value: 758.22, measured value 758.33

λ_(abs) (water): 647 nm, λ_(fl) (water): 671 nm

Example 15 Preparation of Compound 1-15 (1) Compound 10-15

(1.32 g, 20%)

R_(f)=0.52 (RP-C18, acetonitrile/water 3:7 v/v)

¹H NMR (300 MHz, DMSO-d₆): δ 7.79-7.66 (m, 5H), 7.36 (t, 2H, J=12.1 Hz),7.21 (d, 2H, J=7.89 Hz), 6.40 (t, 2H, J=12.8 Hz), 6.14 (dd, 2H, J=3.09Hz, 4.58 Hz), 3.99-3.97 (m, 4H), 2.17 (t, 2H, J=6.23 Hz), 1.83-1.24 (m,19H)

LC/MS, C₃₄H₃₉N₂O₈S₂ ⁻, calculated value: 667.22, measured value: 667.46

λ_(abs) (water): 747 nm, λ_(fl) (water): 784 nm

(2) Compound 1-15

(15.9 mg, 67.3%)

R_(f)=0.52 (RP-C18, acetonitrile/water 3:7 v/v)

¹H NMR (300 MHz, DMSO-d₆): δ 8.08-7.61 (m, 8H), 7.28 (m, 2H), 7.00-6.90(m, 1H), 6.54 (t, 2H, J=12.9 Hz), 6.36 (dd, 2H, J=5.64 Hz, 3.81 Hz),6.25-6.19 (m, 2H), 4.10 (m, 4H), 3.22 (m, 2H), 2.13 (m, 2H, J=7.26 Hz),1.75-1.20 (m, 21H)

LC/MS, C₃₈H₄₆N₃O₉S₃ ⁻, calculated value: 784.24, measured value: 784.34

λ_(abs) (water): 747 nm, λ_(fl) (water): 786 nm

Example 16 Preparation of Compound 1-16 (1) Synthesis of Compound 6B-3

2,3,3-trimethylindolenine (7.96 g, 50 mmol, 1 eq, Aldrich) and6-bromo-n-hexanoic acid (11.7 g, 60 mmol, 1.2 eq, Aldrich) were heatedunder reflux in 50 mL of 1,2-dichlorobenzene for 12 hours. The reactionmixture was allowed to cool to ambient temperature, the solvent wasremoved, and a solid was precipitated through addition of ethyl acetatewas filtered and dried under reduced pressure to obtain a pinkparticulate solid (13.1 g, 73.9%).

R_(f)=0.8 (normal phase, methylene chloride/hexane/methanol 5:1:1 v/v)

¹H NMR (300 MHz, CD₃OD): δ 7.91-7.87 (m, 1H), 7.80-7.79 (m, 1H),7.67-7.69 (m, 2H), 4.56-4.51 (t, J=7.7 Hz, 2H), 2.38-2.33 (t, J=7.7 Hz,2H), 2.03-1.98 (m, 2H), 1.80-1.52 (m, 10H)

(2) Synthesis of Compound 9-13

1,2,3,3-tetramethyl-3H-indoliumiodide (2.00 g, 6.65 mmol, 1 eq, Aldrich)and DPF (1.34 g, 6.863 mmol, 1.03 eq, TCI) were dissolved in 25 mL ofacetic acid and the resulting mixture was heated under reflux for onehour. The reaction mixture was allowed to cool to ambient temperature,filtered and recrystallized two or three times in a solution of ethanoland ether. The resulting orange particles were filtered and dried underreduced pressure (2.31 g, 78%).

R_(f)=0.55 (normal phase, methylene chloride/hexane/methanol 5:1:1 v/v)

¹H NMR (400 MHz, DMSO-d₆): δ 9.27-9.22 (d, J=13.8 Hz, 1H), 7.75-7.49 (m,9H), 5.62-5.59 (d, J=13.8 Hz, 1H), 3.92 (s, 3H), 2.14 (s, 3H), 1.83 (s,6H), 1.71 (s, 3H)

(3) Synthesis of Compound 10-16

The compound 9-13 (2.97 g, 8.4 mmol, 1 eq) and the compound 6b-3 (3.75g, 8.4 mmol, 1 eq) were dissolved in a solution consisting of 300 mL ofethanol and 3 mL of triethylamine and the resulting solution was heatedunder reflux for 30 minutes. The reaction mixture was allowed to cool toambient temperature, distilled under reduced pressure and dried. Theresulting product was purified by normal chromatography using a mixedeluent of dichloromethane, methanol and hexane (5:1:1) to obtain a purecompound 10-16 (2.36 g, 48%).

R_(f)=0.67 (normal phase, methylene chloride/hexane/methanol 5:1:1 v/v)

¹H NMR (400 MHz, DMSO-d₆): δ 8.37-8.30 (dd, J=11.8 Hz, 14.5 Hz, 1H),7.65-7.63 (d, J=6.5 Hz, 2H), 7.46-7.36 (m, 4H), 7.29-7.24 (m, 2H),6.54-6.51 (d, J=13.3 Hz, 2H), 4.09 (m, 2H), 3.64 (s, 3H), 2.32-2.28 (t,2H), 1.69-1.42 (m, 18H)

LC/MS, C₃₀H₃₇N₂O₂ ⁺, calculated value: 457.28, measured value: 457.8

λ_(abs) (methanol): 546 nm, λ_(fl) (methanol): 564 nm

(4) Synthesis of Compound 1-16

A solution of CDI (240 mg, 1.5 mmol, 1.5 eq) in DMF (2.5 mL) was addeddropwise to a solution of the compound 10-16 (460 mg, 1 mmol, 1 eq) inDMF (5 mL). The resulting mixture was stirred for 30 minutes, 160 mg ofHunig's base was added thereto, a solution of2-(2′-chloroethylsulfonyl)ethylamine hydrochloride (210 mg, 1 mmol, 1eq) in 2.5 mL of DMF was added dropwise thereto, and the resultingmixture was stirred at ambient temperature for 24 hours. A solid wasprecipitated through addition of ether, filtered and purified by normalchromatography using a mixed eluent of dichloromethane, methanol andhexane (5:1:1) to obtain a pure compound 1-16 (0.37 g, 52%).

R_(f)=0.68 (normal phase, methylene chloride/hexane/methanol 5:1:1 v/v)

¹H NMR (300 MHz, DMSO-d₆): δ 8.31 (t, 1H), 8.06 (t, J=5.86 Hz, 1H), 7.64(d, J=7.36 Hz, 2H), 7.44 (m, 4H), 7.30 (m, 2H), 6.97 (dd, J=9.93 Hz,9.91 Hz, 1H), 6.48 (d, J=13.5 Hz, 2H), 6.25 (m, 2H), 4.11-4.07 (m, 2H),3.65 (s, 3H), 3.22 (t, J=6.58 Hz, 2H), 2.06 (t, J=7.04 Hz, 2H),1.69-1.42 (m, 20H)

LC/MS, C₃₄H₄₄N₃O₃S⁺, calculated value: 574.31, measured value: 574.22

λ_(abs) (methanol): 546 nm, λ_(fl) (methanol): 564 nm

Example 17 Preparation of Compound 1-17 (1) Synthesis of Compound 9-14

1,2,3,3-tetramethyl-3H-indoliumiodide (1 g, 3.865 mmol, 1 eq) and MDH(1.106 g, 3.672 mmol, 0.95 eq, TCI) were dissolved in a solutionconsisting of 5 mL of acetic acid and 5 mL of anhydrous acetic acid, andthe resulting mixture was heated under reflux for four hours. Thereaction mixture was allowed to cool to ambient temperature, the solventwas removed, and a solid was precipitated by addition of ethyl acetate,filtered, washed with n-butanol several times and dried under reducedpressure (1.56 g, 90%).

R_(f)=0.60 (normal phase, methylene chloride/methanol 5:1 v/v)

¹H NMR (400 MHz, DMSO-d₆): δ 8.87-8.83 (d, J=13.2 Hz, 1H), 8.47-8.44 (t,J=14.9, 1H), 7.75-7.49 (m, 9H), 6.83-6.79 (d, J=14.9, 1H), 5.62-5.59(dt, J=12.9 Hz, 11.5 Hz, 1H), 3.76 (s, 3H), 2.15 (s, 3H), 1.97 (s, 6H),1.66 (s, 3H)

(2) Synthesis of compound 10-17

The compound 9-14 (1 g, 4.49 mmol, 1 eq) was reacted with the compound6b-3 (1.23 g, 10 mmol, 1.35 eq) in the presence of a mixed solvent of 15mL of anhydrous acetic acid and 15 mL of pyridine at 110° C. for 4hours. The reaction mixture was allowed to cool to ambient temperature,and a blue solid was precipitated through addition of ethyl acetate,filtered and dried under reduced pressure. The resulting product waspurified by normal chromatography using a mixed eluent ofdichloromethane, methanol and hexane (5:1:1) to obtain a pure compound10-17 (0.35 g, 13%).

R_(f)=0.70 (normal phase, methylene chloride/hexane/methanol 5:1:1 v/v)

¹H NMR (400 MHz, DMSO-d₆): δ 8.34-8.27 (t, J=14 Hz, 2H), 7.60-7.58 (d,J=8 Hz, 2H), 7.37-7.36 (m, 4H), 7.23 (m, 2H), 6.57-6.51 (t, J=12 Hz,1H), 6.30-6.22 (dd, J=12 Hz, 14 Hz, 2H), 4.07 (bt, 2H), 3.65 (s, 3H),2.18-2.15 (t, J=12 Hz, 2H), 1.95-1.91 (m, 2H), 1.66-1.30 (m, 16H)

LC/MS, C₃₂H₃₉N₂O₂ ⁺, calculated value: 483.3, measured value: 483.11

λ_(abs) (methanol): 641 nm, λ_(fl), (methanol): 678 nm

(3) Synthesis of Compound 1-17

A solution of CDI (73 mg, 0.45 mmol, 1.5 eq) in DMF 0.8 mL was addeddropwise to a solution of the compound 10-17 (150 mg, 0.3 mmol, 1 eq) inDMF 3.5 mL. The resulting mixture was stirred for 30 minutes, 49 mg ofHunig's base was added dropwise thereto, a solution of2-(2′-chloroethylsulfonyl)ethylamine hydrochloride (63 mg, 0.3 mmol, 1eq) in 0.8 mL of DMF was added dropwise thereto and the resultingmixture was stirred at ambient temperature for 24 hours. A solid wasprecipitated through addition of ether and was purified by normalchromatography using a mixed eluent of dichloromethane, methanol andhexane (5:1:1) to obtain a pure compound 1-17 (0.106 g, 49%).

R_(f)=0.67 (normal phase, methylene chloride/hexane/methanol 5:1:1 v/v)

¹H NMR (400 MHz, DMSO-d₆): δ 8.31 (t, J=13 Hz, 2H), 8.02 (m, 1H), 7.60(d, J=7.28 Hz, 2H), 7.36 (d, J=4.48 Hz, 4H), 7.22 (m, 2H), 6.96 (dd,J=10.0 Hz, 9.96 Hz, 1H), 6.55 (t, J=12.2 Hz, 1H), 6.24 (m, 4H), 4.06 (m,2H), 3.68 (s, 3H), 3.20 (t, J=6.68 Hz, 2H), 2.03 (t, J=7.24 Hz, 2H),1.66-1.20 (m, 20H)

LC/MS, C₃₆H₄₆N₃O₃S⁺, calculated value: 600.33, measured value: 600.12.

λ_(abs) (water): 641 nm, λ_(fl) (water): 678 nm

Example 18 Preparation of Compound 1-18 (1) Synthesis of compound 9-15

1,2,3,3-tetramethyl-3H-indoliumiodide (6.626 g, 22 mmol, 1 eq) and GDH(6.265 g, 22 mmol, 1 eq, TCI) were dissolved in 16 mL of anhydrousacetic acid and the resulting solution was allowed to react at 100° C.for one hour. The reaction mixture was allowed to cool to ambienttemperature, a solid was precipitated through addition of distilledwater and was purified, and the residue was washed several times withdistilled water and dried under reduced pressure (5.88 g, 54%).

R_(f)=0.7 (normal phase, methylene chloride/methanol 5:1 v/v)

¹H NMR (400 MHz, CDCl₃): δ 8.19-8.15 (d, J=13.7 Hz, 1H), 7.84-6.89 (m,13H), 5.45-5.39 (dt, J=13.4 Hz, 11.7 Hz, 1H), 4.2 (s, 3H), 1.92 (s, 3H),1.72 (s, 6H), 1.64 (s, 3H)

(2) Synthesis of Compound 10-18

The compound 9-15 (0.5 g, 1 mmol, 1 eq) and the compound 6b-3 (3.54 g, 1mmol, 1 eq) were dissolved in 7 mL of pyridine and the resultingsolution was stirred at 40° C. for 30 minutes. The reaction mixture wasallowed to cool to ambient temperature and the solvent was removed bydistillation under reduced pressure. The residue was purified by normalchromatography using a mixed eluent of dichloromethane, methanol, hexane(5:1:1) to obtain a pure compound 10-18.

(0.175 g, 28%)

R_(f)=0.88 (normal phase, methylene chloride/methanol 5:1 v/v)

¹H NMR (400 MHz, CDCl₃): δ 7.87-7.74 (m, 3H), 7.58-7.55 (d, J=9.0 Hz,2H), 7.40-7.33 (m, 4H), 7.25-7.21 (m, 2H), 6.55-6.49 (m, 2H), 6.35-6.30(d, J=13.9 Hz, 2H), 4.04-4.02 (m, 2H), 3.58 (s, 3H), 2.22-2.17 (t, J=15Hz, 2H), 1.67-1.26 (m, 18H)

LC/MS, C₃₄H₄₁N₂O₂ ⁺, calculated value: 509.32, measured value: 509.25

λ_(abs) (methanol): 740 nm, λ_(fl) (methanol): 775 nm

(3) Synthesis of Compound 1-18

The compound 10-18 (153 mg, 0.3 mmol, 1 eq) was dissolved in DMF (3.5mL) and a solution of CDI (73 mg, 0.45 mmol, 1.5 eq) in DMF (0.8 mL) wasadded dropwise to the solution. The resulting mixture was stirred for 30minutes, 49 mg of Hunig's base was added dropwise thereto, a solution of2-(2′-chloroethylsulfonyl)ethylamine hydrochloride (63 mg, 0.3 mmol, 1eq) in 0.8 mL of DMF was added dropwise thereto, and the resultingmixture was stirred at ambient temperature for 24 hours. A solid wasprecipitated through addition of ether, filtered and purified by normalchromatography using a mixed eluent of dichloromethane, methanol, hexane(5:1:1) to obtain a pure compound 1-18 (0.114 g, 52%).

R_(f)=0.81 (normal phase, methylene chloride/methanol 5:1 v/v)

¹H NMR (400 MHz, CDCl₃): δ 8.03 (t, 1H), 7.87-7.75 (m, 3H), 7.57 (dd,J=2.19 Hz, 2.31 Hz, 2H), 7.38 (m, 4H), 7.22 (m, 2H), 6.97 (dd, J=9.9 Hz,9.9 Hz, 1H), 6.52 (m, 2H), 6.35-6.22 (m, 4H), 4.03 (m, 2H), 3.58 (s,3H), 3.21 (m, 2H), 2.05 (t, J=7.2 Hz, 2H), 1.67-1.26 (m, 20H)

LC/MS, C₃₈H₄₈N₃O₃S⁺, calculated value: 626.34, measured value: 626.38

λ_(abs) (methanol): 741 nm, λ_(fl) (methanol): 773 nm

Examples 19 to 21

The compounds (compounds 1-19 to 21) of examples 19 to 21 were preparedin a manner similar to that used in Examples 1 to 3. The data showingstructures of these compounds are given below:

Example 19 Preparation of Compound 1-19 (1) Compound 9-16

(1.83 g, 73.3%)

R_(f)=0.05 (RP-C18, acetonitrile/water 1:4 v/v)

(2) Compound 10-19

(0.63 g, 17.5%)

R_(f)=0.40 (RP-C18, acetonitrile/water 3:7 v/v)

¹H NMR (300 MHz, DMSO-d₆): δ 8.35 (t, 1H, J=12.2 Hz), 7.79 (s, 2H), 7.65(d, 2H, J=8.37 Hz), 7.39 (d, 2H, J=8.27 Hz), 6.50 (d, 2H, J=13.1 Hz),4.10 (m, 4H), 2.08 (t, 4H), 1.80-1.20 (m, 24H)

LC/MS, C₃₅H₄₃N₂O₁₀S₂ ⁻, calculated value: 715.24, measured value: 715.18

λ_(abs) (water): 551 nm, λ_(fl) (water): 569 nm

(3) Compound 1-19

(98.7 mg, 51.9%)

R_(f)=0.45 (RP-C18, acetonitrile/water 3:7 v/v)

¹H NMR (300 MHz, DMSO-d₆): δ 8.30 (t, 1H, J=13.5 Hz), 8.06-8.00 (m, 2H),7.80 (s, 2H), 7.66 (d, 2H, J=8.22 Hz), 7.39 (d, 2H, J=8.19 Hz), 6.97(dd, 2H, J=9.87 Hz, 9.84 Hz), 6.50 (d, 2H, J=13.3 Hz), 6.24 (m, 4H),4.11 (m, 4H), 3.22 (t, 2H, J=6.42 Hz), 3.09 (m, 2H), 2.06 (m, 4H),1.70-1.14 (m, 28H)

LC/MS, C₄₃H₅₇N₄O₁₂S₄ ⁻, calculated value: 949.29, measured value: 949.32

λ_(abs) (water): 551 nm, λ_(fl) (water): 571 nm

Example 20 Preparation of Compound 1-20 (1) Compound 9-17

(3.96 g, 75.4%)

R_(f)=0.05 (RP-C18, acetonitrile/water 1:4 v/v)

(2) Synthesis of Compound 10-20

(1.15 g, 20.6%)

R_(f)=0.35 (RP-C18, acetonitrile/water 3:7 v/v)

¹H NMR (300 MHz, DMSO-d₆): δ 8.34 (t, 2H, J=12.9 Hz), 7.79 (s, 2H), 7.61(d, 2H, J=8.13 Hz), 7.30 (d, 2H, J=8.28 Hz), 6.64 (t, 1H, J=11.9 Hz),6.27 (d, 2H, J=13.8 Hz), 4.06 (m, 4H), 1.99 (t, 4H, J=6.81 Hz),1.80-1.23 (m, 24H)

LC/MS, C₃₇H₄₅N₂O₁₀S₂ ⁻, calculated value: 741.25, measured value: 741.36

λ_(abs) (water): 649 nm, λ_(fl) (water): 674 nm

(3) Compound 1-20

(47.1 mg, 48.2%)

R_(f)=0.42 (RP-C18, acetonitrile/water 3:7 v/v)

¹H NMR (400 MHz, DMSO-d₆): δ 8.35 (t, 2H, J=13.1 Hz), 8.19-7.99 (m, 2H),7.80 (s, 2H), 7.61 (d, 2H, J=8.20 Hz), 7.31 (d, 2H, J=8.28 Hz), 6.97(dd, 2H, J=10.0 Hz, 9.96 Hz), 6.61 (t, 1H, J=12.4 Hz), 6.31-6.21 (m,4H), 4.07 (m, 4H), 3.22 (m, 2H), 3.13 (m, 2H), 2.18 (t, 2H, J=7.20 Hz),2.03 (t, 2H, J=7.28 Hz), 1.67-1.20 (m, 28H)

LC/MS, C₄₅H₅₉N₄O₁₂S₄ ⁻, calculated value: 975.3, measured value: 975.47

λ_(abs) (water): 649 nm, λ_(fl) (water): 675 nm

Example 21 Preparation of Compound 1-21 (1) Compound 9-18

(5.82 g, 99%)

R_(f)=0.3 (RP-C18, acetonitrile/water 1:2 v/v)

(2) Compound 10-21

(1.26 g, 33%)

R_(f)=0.32 (RP-C18, acetonitrile/water 3:7 v/v)

¹H NMR (300 MHz, DMSO-d₆): δ 7.70-7.62 (m, 6H), 7.28 (t, 1H, J=12.8 Hz),7.14 (d, 2H, J=8.38 Hz), 6.28 (t, 2H, J=12.6 Hz), 6.02 (d, 2H, J=13.6Hz), 3.90 (m, 4H), 2.12 (t, 4H, J=7.28 Hz), 1.69-1.31 (m, 24H)

LC/MS, C₃₉H₄₇N₂O₁₀S₂ ⁻, calculated value: 767.27, measured value: 767.44

λ_(abs) (water): 749 nm, λ_(fl) (water): 786 nm

(3) Compound 1-21

(42.3 mg, 42.2%)

R_(f)=0.38 (RP-C18, acetonitrile/water 3:7 v/v)

¹H NMR (300 MHz, DMSO-d₆): δ 8.01 (t, 2H, J=5.68 Hz), 7.86 (t, 2H,J=13.1 Hz), 7.75-7.73 (m, 3H), 7.61 (d, 2H, J=8.20 Hz), 7.28 (d, 2H,J=8.48 Hz), 6.97 (dd, 2H, J=9.96 Hz, 9.92 Hz), 6.54 (t, 2H, J=11.9 Hz),6.34 (d, 2H, J=13.6 Hz), 6.26-6.22 (m, 4H), 4.11 (m, 4H), 3.21 (t, 4H,J=6.80 Hz), 2.03 (t, 4H, J=7.24 Hz), 1.62-1.23 (m, 28H)

LC/MS, C₄₇H₆₁N₄O₁₂S₄ ⁻, calculated value: 1001.32, measured value:1001.50

λ_(abs) (water): 750 nm, λ_(fl) (water): 792 nm

Example 22 Testing of Binding Force to Amine Compound

A dilution of 0.1306 μmol of benzyl amine (molecular weight: 107.16,Aldrich) in 10 μl of DMF was added to a 0.1M phosphate buffer solution(pH 5, 20 μl), and 1 μl of a solution of the compound 1-16 (1 mg) in DMFwas mixed with the resulting solution. Similarly, a solution of benzylamine and the compound 1-16 was prepared using the phosphate buffersolution (pH 6, 7, 7.5, 8, 8.5, 9, 9.5 and 10) and the resultingsolution was allowed to react at 40° C. for 30 minutes. It is estimatedthat reaction of the benzyl amine with the compound 1-16 causesproduction of a compound having the structure represented by Formula 13below:

After reaction, retention time (RT) of benzyl amine, the compound 1-16and the product (compound 13) were confirmed by HPLC to obtain areaction yield. At this time, methanol was used as a mobile phase, flowrate was 2.5 mL/min and analysis time was 80 minutes, and multi-scanningwas carried out at wavelengths of 254, 365, 450, 550 and 650 nm. FIG. 1shows absorbance at a wavelength of 550 nm. In FIG. 1, “▪” is a valueanalyzed from the reaction product stored at 4° C. for one day, and “”is a value obtained from the reaction product 7 days after theafore-mentioned analysis. As can be seen from FIG. 1, the reaction yieldis 60% or higher in the range of pH 8.5 to 9.0, and the reaction yieldis 80% or higher in the range of pH 8.5 to 9.0, since the reaction isslow even at a low temperature for a long time. Accordingly, thecompound 1-16 of the present invention exhibits superior stability andconsiderably excellent binding force to the amine compound.

Example 23 Protein Staining Test (1)

Each vial of one pack of Amershame™ LMW calibration kit (17-0446-01) forSDS electrophoresis commercially available from GE healthcare Co., Ltd.contained six kinds of marker proteins (576 μg), so-called,phosphorylase b (97 kD, 67 μg), albumin (66 kD, 83 μg), ovalbumin (45kD, 147 μg), carbonic anhydrase (30 kD, 83 μg), a trypsin inhibitor(20.1 kD, 80 μg), and α-lactalbumin (14.4 kD, 116 μg).

A phosphate buffer solution (250 μl, 0.1 M) was added to one vialcontaining the marker proteins at ambient temperature (20° C.) and wasaliquoted in an amount of 25 μl to four e-tubes (25 μg protein/25 μlbuffer, 6.9×10⁻⁵ μmol in 25 μl buffer solution).

The compound 1-1 (1 mg) was dissolved in 100 μl of DMF, 1 μl of thesolution was placed in the afore-mentioned e-tube, and the resultingmixture was homogeneously mixed using a vortex shaker and a centrifuge.The e-tube was placed in a heating block set to 30° C. and the reactionwas proceeded. After one hour, one e-tube was collected and stored at−20° C. The reaction of one e-tube was finished after one, two and 16hours. Finally, the reaction times were adjusted to one, two, four and20 hours.

In addition, the solution of the compound 1-1 was collected in an amountof 0.25 μl, and reacted in the same manner as in the reaction of thesolution of 1 μl.

The reaction product of the solution (1 μl) of the compound 1-1 and thereaction product of the solution (0.25 μl) of the compound 1-1 wereloaded in an amount of 15 μA and separated via gel electrophoresis. Theresults thus obtained are shown in FIGS. 2A and 2B. Electrophoresis wascarried out at 125V for two hours.

FIG. 2A is an image observed by the naked eye and FIG. 2B is afluorescent image. In FIG. 2, {circle around (1)} to {circle around (4)}are obtained from 1 μl of a dye solution and {circle around (5)} to{circle around (8)} are obtained from 0.25 μl of a dye solution. As canbe seen from FIG. 2, as an amount of the compound used increases,protein lanes appear clearer, and as staining time increases, thestaining is more efficient and various proteins are homogeneouslystained.

Example 24 Protein Staining Test (2)

Phosphate buffer solutions (pH 5, 6, 7, 7.5, 8, 8.5, 9 and 10) wereprepared, the reaction was carried out using the same marker proteinsand the same compound (compound I-1) as in example 23, and using all ofthe dye compound solutions in an amount of 0.5 μl for 2 hours.

Gel electrophoresis was carried out in the same manner as in example 23,and an image observed by the naked eye (FIG. 3A) and a fluorescent imageobtained using a Geliance 600 (FIG. 3B) are shown in FIG. 3. As can beseen from FIG. 3, proteins were stained very little at pH 5, whereasstained proteins were observed by both the naked eye and fluorescenceanalysis in the overall pH range and in particular, proteins whichreacted with buffer solutions (pH 8.5 to 10) exhibited the strongestfluorescence.

Example 25 Protein Staining Test (3)

Each vial of one pack of Amershame™ HMW calibration kit (17-0445-01) fornative electrophoresis (GE healthcare Co., Ltd.) contained five markerproteins (250 μg), that is, thyroglobulin (665 kD, 76 μg (30.4%)),ferritin (440 kD, 50 μg (20%), catalase (232 kD, 36 μg (14.4%), lactatedehydrogenase (140 kD, 48 μg (19.2%)), and bovine serum albumin (67 kD,40 μg (16%)). The proteins were dissolved in 500 μl of water in eachvial and filtered using VIVASPIN 500 (10 kD, Sartorius) and filteringwas repeated several times with addition of water, to completely removematerials except the proteins. A 1 μl aliquot of the residue was placedin each of ten e-tubes.

Phosphate buffer solutions, carbonate buffer solutions and tris buffersolutions in concentrations of 0.1 M, 0.01 M and 0.001 M were prepared,a total of the 9 buffer solutions with various concentrations were addedin an amount of 18 μl to the previously-prepared e-tubes, and the samevolume of distilled water was added to the one remaining e-tube.

The compound 1-1 (1 mg) was dissolved in 100 μl of DMF, 1 μl of thesolution was collected and added to the previously prepared 10 e-tubes,and the resulting mixtures were homogeneously mixed using a vortexshaker and centrifuge.

The e-tubes were added to the heating block set to 36.5° C. and thereaction was allowed to proceed for 4 hours. Gel electrophoresis wascarried out in the same manner as in example 23. An image observed bythe naked eye (FIG. 4A) and a fluorescence image obtained using aGeliance 600 (FIG. 4B) are shown in FIG. 4. Marker proteins (GEhealthcare) used herein were developed for SDS-PAGE applications, butstaining thereof may be confirmed, although they can be accuratelyseparated depending on sizes. In each image, ten lanes, that is, 0.1 M,0.01M, and 0.001M phosphate buffer solutions 0.1 M, 0.01M and 0.001Mcarbonate buffer solutions, and 0.1 M, 0.01M and 0.001M tris-buffersolutions are arranged from the left to the right in this order, and therightmost lane is obtained by reaction in water. Proteins reacted inwater are generally stained well and, from the fluorescence image, itcan be seen that the 0.01M phosphate and carbonate buffer solutionsexhibited the most efficient expression, in particular, and 0.01 Mphosphate buffer solution exhibited the most superior fluorescence.

Comparative Test (1)

1 mg of Amershame™ Cy™5 Mono NHS Ester (PA15101, GE Cy5, GE healthcare)and 1 mg of the compound 1-2 were dissolved in 100 μl of DMF, and thesolution was tested in the same manner as in example 22. GE Cy5 and thecompound 1-2 have different molecular weights. For this reason, when thecompound was aliquoted in an amount of 1 the amount of the GE dyecorresponds to 0.01376 μmol, and the amount of the compound 1-2corresponds to 0.01339 μmol. Dilutions of equivalent moles of benzylamine, benzyl alcohol (molecular weight: 108.14, Aldrich) and benzylmercaptan (molecular weight: 124.20, Aldrich) in 20 μl of DMF wereprepared.

6 sets, each including 12 phosphate buffer solutions (0.1 M, 229 μl)having different pHs(i.e., pH 5, 6, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 11.5and 12) were prepared. One set included 1 μl of GE Cy5 dye solution and20 μl of benzyl amine. In the same manner, the benzyl alcohol and benzylmercaptan were mixed with the GE Cy5 dye solution. In the same manner,the solution of the compound 1-2 and three types of benzyl compoundswere reacted with three sets of phosphate buffer solutions. 64 e-tubesthus prepared were reacted in a heating block at 36.5° C. for 4 hours.

Formula 14 below has a structure represented by the compound wherein thedye of GE Cy5 is bound to benzyl amine.

LC/MS, C₄₀H₄₆N₃O₇S₂ ⁻, calculated value: 744.28, measured value: 744.37

Meanwhile, Formulae 15 to 17 below have structures represented bycompounds wherein the compound 1-2 of the present invention is bound tobenzyl amine, benzyl alcohol and benzyl mercaptan.

LC/MS, C₄₄H₅₅N₄O₉S₃ ⁻, calculated value: 879.31, measured value: 879.57

LC/MS, C₄₄H₅₄N₃O₁₀S₃ ⁻, calculated value: 880.3, measured value: 880.53

LC/MS, C₄₄H₅₅N₃O₉S₄, calculated value: 897.28, measured value: 897.03

Retention time (RT) of benzyl amine, the compound 1-16 and the product(compound 13) were confirmed by HPLC to obtain a reaction yield. At thistime, a 20% acetonitrile solution was used as a mobile phase, flow ratewas 1 mL/min and analysis time was 30 minutes, and multi-scanning wascarried out at wavelengths of 254, 365, 600, 650 and 700 nm.

The reaction yield, based on absorbance at a wavelength of 650 nm, isshown in FIGS. 5A to 5C.

As shown in 5A to 5C, the compound 1-2 of the present invention reactswith benzyl amine, benzyl alcohol and benzyl mercaptan at a high yield,in particular, exhibited a reaction yield of about 90% in the range ofpH 9 to 11. GE Cy5 dye reacts only with benzyl amine, to obtain a yieldof 50% in the range of pH 9 to 11, whereas it does not react with benzylalcohol or benzyl mercaptan. These results can be supported by the factthat, when the molecular weight was measured by LC/MS, the molecularweight of the structure wherein the dye of GE Cy5 is bound to benzylalcohol or benzyl mercaptan was not obtained. These test resultsindicate that the compound of Formula 1 exhibits superior binding forceto a compound containing amine, hydroxyl or thiol groups.

Comparative Test (2)

One tube containing protein molecular weight marker (Broad, Code No.3452 available from Takara Bio Inc.) comprises proteins (18 μg/μl, totalvolume of 50 μl), a tris buffer solution, EDTA, NaCl and glycerol. Theproteins include 9 kinds of proteins, namely, myosin (200 kD),β-galactosidase (116 kD), phosphorylase B (97.2 kD), serum albumin (66.4kD), ovalbumin (44.3 kD), carbonic anhydrase (29 kD), a trypsininhibitor (20.1 kD), lysozyme (14.3 kD) and aprotinin (6.5 kD). Theprotein aqueous solution (available from Takara Bio Inc.) was filteredusing VIVASPIN 500 (5kD, Sartorius), and a 1 μl aliquot of the resultingfiltrate was placed in each of ten e-tubes.

18 μl of a phosphate buffer solution (pH 8.5 0.1 M) was placed in thepreviously prepared 10 e-tubes. For the five e-tubes, 1 mg of Amershame™Cy™3 Mono NHS Ester available from GE healthcare (PA13101, hereinafterreferred to as “GE Cy3”) and 1 mg of the compound 1-1 were dissolved in100 μl of DMF. The solution of GE Cy3 and the compound 1-1 werealiquoted in amounts of 0.25 μl, 0.125 μl, 0.0625 0.025 μl and 0.0025and DMF was added to adjust the final volume of these solutions to 1 μlsuch that the volumes of these solutions were equivalent. The prepared 5dye solutions were placed in the 10 e-tubes containing the previouslyprepared proteins, and the resulting mixture was homogeneously mixedusing a vortex shaker and a centrifuge. Each e-tube was reacted in aheating block at 36.5° C. for 4 hours.

Gel electrophoresis was carried out in the same manner as in Example 23,an image observed by the naked eye (FIG. 6A), and a fluorescence imageobtained using a Geliance 600 (FIG. 6B) are shown in FIG. 6. In theimages, references, GA to GE are obtained from the GE Cy3 dye, A to Eare obtained from the compound 1-1 of the present invention, and 6^(th)to 12^(th) lanes of a gel image are color size markers. (Takara,Prosieve Color Protein Marker, Cat. No. 50550). As can be seen from FIG.6, for the total 9 proteins, the compound 1-1 of the present inventionexhibited color and fluorescence intensities, which can be identified bythe naked eye, superior to or comparable to the GE Cy3 dye. In addition,the compound 1-1 exhibited more considerable fluorescence performance atlow dye concentrations, as compared to the dye of GE.

For more detailed comparison, the fluorescence intensity programGelTools (ver.3.07.14) installed in the Geliance 600 apparatus was usedto compare 9 proteins, and the results thus obtained are shown in FIGS.7A to 7I. In FIGS. 7A to 7I, “GE” is obtained from the GE dye, and“KIST” is obtained from the compound 1-1. As shown in FIGS. 7A to 7I,the compound 1-1 exhibited superior fluorescence performance for all 9proteins, as compared to the dye marker protein of GE. In particular,difference in fluorescence performance increases, as the molecularweight of the compound 1-1 increases to 50 kD or higher, which meansthat the compound of the present invention exhibits superiorhigh-molecular compound staining performance.

Comparative Test (3)

Each tube of prosieve protein markers (Code No. 50547) available fromTakara Bio Inc. contained proteins (at a concentration of 550 μg/500 μl,total volume of 500 μl), a tris-buffer solution, EDTA, NaCl andglycerol. Although the kinds of proteins contained in each prosieveprotein marker are not mentioned in detail, 10 kinds of proteins havingmolecular weights of 225 kD, 150 kD, 100 kD, 75 kD, 50 kD, 35 kD, 25 kD,15 kD, 10 kD and 5 kD, wherein the protein having a molecular weight of50 kD is present in an amount of 100 μg and the remaining proteins werepresent in an amount of 50 μg, are known. This protein solution wasfiltered using a VIVASPIN 500 (10 kD, Sartorius) and the filtering wasrepeated several times with addition of 500 μl of water, to remove thematerials except the proteins. A 2 μl aliquot of the residue was placedin each of 12 e-tubes.

1 mg of the product (dye available from GE health care) used inComparative Example 2 (hereinafter, referred to as “GE Cy3”), 1 mg ofthe product used in Comparative Example 1 (hereinafter, referred to as“GE Cy5”), and 1 mg of the product, Amershame™ Cy™7 Mono NHS Ester(PA17101, hereinafter, referred to as “GE Cy7”) were dissolved in 100 μlof DMF.

Meanwhile, 1 mg of the compounds 1-7, 1-1, 1-4, 1-10, 1-13, 1-19, 1-16,1-2 and 1-3 was dissolved in 100 μl of DMF, to prepare a solution ofcompound 1-7 (hereinafter, referred to as “3-1”), a solution of thecompound 1-1 (hereinafter, referred to as “3-2”), a solution of thecompound 1-4 (hereinafter, referred to as “3-3”), a solution of thecompound 1-10 (hereinafter, referred to as “3-4”), a solution of thecompound 1-13 (hereinafter, referred to as “3-5”), a solution of thecompound 1-19 (hereinafter, referred to as “3-6”), a solution of thecompound I-16 (hereinafter, referred to as “3-7”), a solution of thecompound 1-2 (hereinafter, referred to as “5-1”) and a solution of thecompound 1-3 (hereinafter, referred to as “7-1”).

18 μl of a phosphate buffer solution (pH 9.5, 0.1 M) was placed in eachof the previously prepared 12 e-tubes, 1 μl of GE Cy3, 3-1, 3-2, 3-3,3-4, 3-5, 3-6, 3-7, GE Cy5, 5-1, GE Cy7 and 7-1 was placed in therespective e-tubes and the resulting solutions were homogeneously mixedusing a vortex shaker and a centrifuge. The resulting mixture wasreacted in a heating block at 36.5° C. for 6 hours.

Gel electrophoresis was carried out as in Example 23 and an imageobserved by the naked eye (FIG. 8A), and a fluorescence image measuredusing a Geliance 600 (FIG. 8B) are shown in FIG. 8. FIGS. 8A and 8B showcolor and fluorescence of GE Cy3-, 3-1-, 3-2-, 3-3-, 3-4-, 3-5-, 3-6-,3-7-, GE Cy5-, 5-1-, GE Cy7- and 7-1-marked proteins from left to right,respectively. 3-2, 3-3 and 3-4 expressed considerably superiorfluorescence, which means that proteins were marked with a larger amountof dye, as compared to GE Cy3. GE Cy5 and 5-1 exhibited substantiallyidentical fluorescence, GE Cy7 and 7-1 had absorbance or fluorescencewavelengths in the range of near infrared rays, and thus cannot beobserved by the naked eye, but exhibit substantially identicalfluorescence performance.

As apparent from the afore-going, the present invention enablesbiomolecular researchers to handle dyes more stably, when performinglabeling tests. In particular, binding of biomolecules to dyes does notcause by-products, thus eliminating the necessity of additionalpurification processes. In addition, the compound of the presentinvention enables easy long-term storage due to superior stability andis more applicable to users who stain large molecular-weightbiomolecules for a long period and handle more complicated biomolecules.

Although the preferred embodiments of the present invention have beendisclosed for illustrative purposes, those skilled in the art willappreciate that various modifications, additions and substitutions arepossible, without departing from the scope and spirit of the inventionas disclosed in the accompanying claims.

1. A cyanine compound represented by Formula 1 below:

wherein R₁ and R_(1′) are each independently hydrogen, a sulfonic acidgroup or a sulfonic acid base; R₂, R_(2′), R₃ and R_(3′) are eachindependently hydrogen or a C₁-C₆ alkyl group; R₄ is hydrogen, a C₁-C₆alkyl group, a carboxyl group, —CONH(CH₂)_(L)SO₂CH═CF₂,—CONH-para-(C₆H₄)SO₂CH═CH₂ or —CONH-meta-(C₆H₄)SO₂CH═CH₂; B is(CH₂)_(l), para-(C₆H₄) or meta-(C₆H₄); m and m′ are each independentlyan integer of 1 to 5; and L, l and n are each independently an integerof 1 to
 5. 2. The compound according to claim 1, wherein the compound isselected from the group consisting of:


3. The compound according to claim 1, wherein the compound fluoresces ata wavelength of 500 to 800 nm.
 4. The compound according to claim 1,wherein the compound labels a biomolecule, a nanoparticle or an organiccompound containing an amine group, a hydroxyl group or a thiol group.5. The compound according to claim 4, wherein the biomolecule isselected from the group consisting of proteins, peptides, carbohydrates,sugars, lipids, antibodies, proteoglycans, glycoproteins and siRNA.
 6. Amethod for labeling biomolecules, nanoparticles, or organic compoundscontaining an amine group, a hydroxyl group or a thiol group with thecompound of Formula 1 according to claim
 1. 7. The method according toclaim 6, wherein the labeling is carried out by binding vinyl sulfonepresent in the compound of Formula 1 to the amine, hydroxyl or thiolgroup present in the biomolecules, nanoparticles or organic compoundsthrough reaction of the vinyl sulfone with the amine, hydroxyl or thiolgroup.
 8. The method according to claim 6, wherein the biomolecule isselected from the group consisting of proteins, peptides, carbohydrates,sugars, lipids, antibodies, proteoglycans, glycoproteins and siRNA. 9.The method according to claim 6, wherein the solvent for labeling is abuffer solution selected from the group consisting of a phosphate buffersolution, a carbonate buffer solution and a tris buffer solution, anorganic solvent selected from the group consisting of dimethylsulfoxide, dimethyl formamide, methanol, ethanol and acetonitrile, orwater.
 10. The method according to claim 6, wherein the labeling iscarried out at pH 5 to
 12. 11. The method according to claim 6, whereinthe labeling is carried out by reacting the compound of Formula 1 withbiomolecules, nanoparticles or organic compounds at a temperature of 20to 80° C. for 30 minutes to 48 hours.
 12. A material selected frombiomolecules, nanoparticles and organic compounds labeled with thecompound of Formula 1 according to claim
 1. 13. The material accordingto claim 12, wherein the biomolecule is selected from the groupconsisting of proteins, peptides, carbohydrates, sugars, lipids,antibodies, proteoglycans, glycoproteins and siRNA.
 14. A compoundrepresented by Formula 10, as an intermediate of the compound of Formula1 according to claim 1:

wherein R₁ and R_(1′) are each independently hydrogen, a sulfonic acidgroup or a sulfonic acid base; R₂, R_(2′), R₃ and R_(3′) are eachindependently hydrogen or a C₁-C₆ alkyl group; R₄ is hydrogen, a C₁-C₆alkyl group or a carboxyl group; and m, m′ and n are each independentlyan integer of 1 to
 5. 15. The compound according to claim 14, whereinthe compound is selected from the group consisting of:


16. A method for preparing a compound of Formula 1, comprising: reactinga compound of Formula 4 with a compound of Formula 5 or 7, to obtain acompound of Formula 6a;

reacting the compound of Formula 6a with a compound of Formula 8 toobtain a compound of Formula 9;

reacting the compound of Formula 9 with a compound of Formula 6b toobtain a compound represented by Formula 10;

reacting the compound of Formula 10 with 1,1′-carbonyl diimidazole orN,N-disuccinimidyl carbonate to obtain a compound of Formula 11a or 11b;and

reacting the compound of Formula 11a or 11b with a compound representedby Formula 12 in the presence of a Hunig's base to obtain the compoundof Formula
 1.

wherein R₁ and R_(1′) are each independently hydrogen, a sulfonic acidgroup or a sulfonic acid base; R₂, R_(2′), R₃ and R_(3′) are eachindependently hydrogen or a C₁-C₆ alkyl group; R₄ is hydrogen, a C₁-C₆alkyl group, a carboxyl group, —CONH(CH₂)_(L)SO₂CH═CH₂,—CONH-para-(C₆H₄)SO₂CH═CH₂ or —CONH-meta-(C₆H₄)SO₂CH═CH₂; R₅ is ahalogen atom selected from the group consisting of fluorine, chlorine,bromine and iodine, or a sulfato group (—OSO₃H); A is hydrogen or anacetyl group; B is (CH₂)_(l), para-(C₆H₄) or meta-(C₆H₄); X is a halogenatom selected from the group consisting of fluorine, chlorine, bromineand iodine; m and m′ are each independently an integer of 1 to 5; and L,l and n are each independently an integer of 1 to 5.